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  1. May 2019
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    1. A microtipful of cellsfor each yeaststrainfrom appropriate mediumwassuspended in 10μlzymolyase cocktailandincubated at 37ºC for 90 min. 2 μlof zymolyase-treated cell suspension was used as template in 25 μlPCR reaction
    2. Yeast colony PCR
    3. Alipophilic styryl dye,FM4-64,is a vital stain which istakenupby cells viaendocytosis through plasma membrane(Vida and Emr, 1995). Therefore, it fluorescesonly in live cells. Importantly, neitherfixed cells canbe stained with FM 4-64norcells canbe fixed afterFM 4-64staining. For vacuole staining, single colony of the test strain grown onYPD plate was inoculated in 10 ml YPD medium for overnight. 100 μlovernight culture was inoculated in fresh YPD medium and incubated at 30ºC for 3 hto obtain log-phase cells. C. glabratacells from 1 ml log-phase culture were harvested at 4,000 rpm for 5 minin a table top centrifuge. Supernatant was aspirated out,cells were resuspended in 50 μl YPD medium and 1 μl FM 4-64 (16 μM final concentration) was added.C. glabratacells were incubated in a 30ºC water bath for 30 min. 1 mlYPD medium was added and cells were harvested at 4,000 rpm for 5 minin a table-top centrifuge. After discarding supernatant,C. glabratacells were washed with fresh YPD medium and resuspended in 1 ml YPD medium. C. glabratacells were incubated at 30ºC for 90 min, washed with 1 mlsterile water and were resuspended in 50 μl YNB medium. Labeled C. glabratacells were observed underfluorescence microscope in red filter(730nm)
    4. Stainingof C. glabratavacuoleswith FM4-64
    5. centrifugation at 5,000 rpm for 4 minat room temperature. Harvested cells werewashed with PBS and treated with different compoundse.g.H2O2. After treatment,cells were harvested and further processed according to the type of experiments performed
    6. For several experiments, log-phase C. glabratacells were harvested and treated with different compounds. For this, single colony of aC. glabratastrain was inoculated in YPD-liquid medium and grown for 14-16 h at 30ºC withcontinuous shaking at 200 rpm. Overnight cultures were reinoculated in YPD medium to an initial OD600of 0.1 andgrown for another 4 h. These log-phase cells were harvested by
    7. Harvesting of and treatment to logarithmic phase C. glabratacells
    8. For opsonization,C. glabratacells were incubatedwith 1 μg/μl human IgG for 30 min at 37°C and washed thrice with PBS. Alternatively, yeast cells were incubated with 25% human serum at 37°C for 30 min followed by threePBS washes
    9. Opsonizationof C. glabratacells
    10. C. glabratastrains were grown overnight in YPD medium. Cellswereharvested from 1 mlcultureandwashed withPBS.Cells were next washed with50mM NaH2PO4andresuspendedin 100 μlFITC-dextran(50mg/ml). After incubation at 37°Cfor 45 min, cells were washed thrice with PBS for complete removal of FITC-dextran.Yeast cells were resuspended in 1 ml PBS and used to infect PMA-treated THP-1 cells in 4-chambered glass slide
    11. Fluorescein isothiocyanate(FITC)staining of C. glabratacells
    12. 24 h post infection, THP-1 macrophages were washed thrice with PBS, lysed in water and recovered yeast cells were used to infect THP-1 cells at a MOIof 1:10. Three rounds of macrophage infection foreach mutant pool were carried out to enrich for the desired mutants in the final population. The lysate of 3rdround infection was inoculated in YPD medium for overnight (output). Cells were harvested, genomic DNA isolated from each input and output cell pellet andunique signature tags were PCR-amplified with P32-labeledα-dCTP using primers complementary to theinvariant region flanking each unique tag sequence. LabeledPCR products were denatured at 95°C for 10 min, chilled on ice and were hybridized tonylon membranescarrying immobilized plasmid DNA containing 96 unique tagsfor 14-16 h at 42°C.Membranes were washed twicewith 0.1X SSC bufferand exposed to phosphorimager screen for 2-4 h. Radioactive counts for each spot were quantified using Image Quant and Fuji Multi Gauge V3.0 software. Relative percentage intensity for individual spot was calculated with respect to allspots present oneach hybridizedmembrane
    13. YPD-grown cultures (0.05 OD600) of each mutant pool (96 mutants, each carrying a unique signature tag) were either inoculated in YPD medium for overnight (input) or used to infect differentiated THP-1 cells (1X106). After 2 h incubation, non-cell-associated yeastcellswere removed by washing THP-1 cellsthricewith PBS. At
    14. Screening of C. glabrataTn7insertion mutant library
    15. Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days
    16. Yeast transformation
    17. Identified mutants were phenotypically characterized in 96-well plate format. Mutant cultures were grown in YPD medium for overnight, diluted 150-fold in PBS and 5 μl of cell suspension was spotted on different plates with a 96-pin replicator. Growth was recorded after 1-2 daysof incubation at 30°C
    18. Phenotypic profiling
    19. at 30°C andimages were captured after 2-8daysof incubationdepending upon the medium used
    20. Yeast strains were grown in YPD medium for 14-16 hat 30°Cunder continuous shaking at 200 rpm. Cells were harvested from 1 mlculture, washed with PBS and were diluted to an OD600of 1. Five ten-fold serial dilutions were preparedfrom aninitial culture of 1OD600.4 μl cultureof each dilution was spotted onYNB-agar plates containing different carbon sources. For spotting on YPD plates containing different compounds, 3 μl cultureof each dilution was spotted. Plates were incubated
    21. Serial dilution spotting assay
    22. For growth analysis of a C. glabratastrain,single colony wasinoculated in appropriate broth medium and grown for 14-16 h. Overnight grown culture was used to inoculate the test medium toan initial OD600of 0.1-0.3. Cultures were transferred to a shaker incubator set at 30°C and 200 rpm. Absorbance ofcultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at 600 nm at regular time-intervalstill 48h. Absorbancevalues were plotted with respect to time and generation time was determined from the logarithmic (log)phase of cell growth usingthefollowing formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf= Number of cells at time T2(1 OD600of C. glabrata corresponds to 2 X 107cells.)Ni= Number of cells at time T1(calculated from OD600value as mentioned above)
    23. Growth analysis and determination of generation time
    24. Bacterial strainEscherichia coli DH5αused for cloning purposewas revived on LB medium and grown at 37°C withcontinuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to growbacterial strains carrying plasmids. AnotherE. coli strain,BW23473,was used to rescue the Tn7transposon cassette from C. glabrataTn7insertion mutants. For plasmid DNA purification, bacterial strains were grown overnight in LB broth medium containingsuitable antibiotics
    25. C. glabratastrains were routinely grown either in rich YPD medium or synthetically-defined YNB medium at 30°C withcontinuous shaking at 200 rpm unless otherwise stated. In general, C. glabratafrozen glycerol stocks wererevivedonYPD medium by streaking and allowed to grow for 1-2 days. C. glabratastrainsharboringthe plasmid with URA3as selectable marker were revived onCAA medium.To prepare liquid cell culture, single colony of eachC. glabratastrainwasinoculated either in YPD or YNB broth mediumand grown for 14-16 h. C. glabratastrains streaked on plates were storedat 4°C fora maximum period of2 weeks
    26. Strains and culture conditions
    27. Microbiological methods
    28. To collectmacrophage-internalized yeast cellsfor RNA and protein extraction, 107THP-1 monocytes were seeded in 100 mm cell culture dishes and treated with PMA. PMA-differentiated THP-1 macrophages were infected with appropriateC. glabratastrainsto a MOIof 1:1. Equal numberof C. glabratacells wasinoculated inRPMI medium as control. Two hourspost infection,non-phagocytosed yeast cells were removed by washing THP-1 macrophages thrice with PBS. At different time points, culture dishes were washed twice with chilled PBS and 2 mlchilled sterile water was added toeach dish to lyse the macrophages. Corresponding cultures grown in RPMI medium were transferred to50 ml polypropylene tubesand transferred on ice. Lysates were collected by scrapping the macrophage monolayer and transferred to50 ml polypropylene tubes.RPMI-grown and macrophage-internalized C. glabratacells were harvested by centrifugation at 2,500g for 8 min. Macrophage cell debris were removed frommacrophage-internalized cells by repeated washing with chilled sterile water. Harvested C. glabratacells were stored at -20ºC till further use
    29. Harvesting of macrophage-internalized C. glabratacellsfor RNA and protein extraction
    30. undertissueculture conditionsfor 45-60min andfixed in 3.7% formaldehydeas described earlier.For DAPI staining, Vectashield mounting medium containing DAPI was used and slides were visualized under confocal microscope.For heat killing, yeast cells were harvested from 1 ml culture, washed, resuspended inPBS andwere incubated at 95°C for 5 min
    31. PMA-treated THP-1 macrophages were infected with C. glabratacells to a MOIof 1:1 in four-chambered slides and incubated at 37°C and 5%CO2. After 1 hcoincubation, each chamber was washed thrice with PBS to eliminate extracellular yeast cellsand medium was replaced with fresh prewarmed RPMI medium containing100 nM Lysotracker Red DND-99.Infected THP-1 macrophageswere incubated
    32. Lysotracker staining
    33. For confocal microscopyanalysis, 5X105THP-1 cells were seeded and treatedwithPMA in 4-chambered slides. Differentiated THP-1 macrophageswere infected either with FITC-labeled or GFP-expressingC.glabratastrains to a MOIof 1:1. At different time intervals, medium was aspirated out from each chamber of 4-chambered slides and chamberes were washed twice with PBS. To fixthe infected macrophages,500 μlformaldehyde(3.7%) was added gently toeach chamber andincubated for 15 minat room temperature. Each chamber of the slide was washed twice withPBS to remove formaldehyde solution completely. To permeabilize the fixed cells, 500 μl Triton-X (0.7%) was dispensed toeach chamber and slide wasincubated at room temperature for 5 min. Chambers of the slide werewashed twice with PBS, separated from the slideusing a chamber removal device andwere air dried. Coverslips were placed onslides using Vectashield mounting mediumand bordersweresealed withnail paint. Slides werestored at 4°C until used forfluorescence imaging
    34. Fixing of PMA-treated THP-1 macrophages
    35. THP-1 cells were seeded ina 24-well tissue culture plate to a celldensity of 1 million cells per well,treated with PMA and were infected with yeast cells to a MOIof 10:1. Two hours post infection, cells were washed thrice with PBS and medium was replacedwith fresh prewarmed RPMI medium.Plates wereincubatedat 37ºCfor 24 h. Supernatants were collected,centrifuged at 3,000 rpm for 5 minto get rid of particulate matter,if any, andwerestored at -20°C until use. Estimation of different cytokines wasperformed using BD OptEA ELISA kits as per the supplier’s instructions
    36. Cytokines measurement
    37. Forinfection of THP-1 cells with single C. glabratastrain, PMA-treatedTHP-1 monocytes were seeded in 24 wellcell culture plate toa seeding density of 1 million cells per well. To prepare C. glabratacells for macrophage infection, single colony of the desiredstrain wasinoculated in YPD medium and allowed to grow for 14-16 hat 30°C. C. glabratacellsfrom 1ml overnight culture were harvested, washed with PBS andcell density was adjustedto 2X107cells/ml.50 μl of thisC. glabratacell suspension wasinfectedto macrophages to a MOIof 10:1. Two hours post infection, infected THP-1 macrophages were washed thrice with PBS to removenon-phagocytosed yeast cells and medium was replacedwith fresh prewarmed medium. Atdifferent time points post infection,infected THP-1 macrophages were washed with PBS three timesandlysed in 1 mlsterilewater. Lysates were collected by scrapping the wells with a micropipette tip, diluted in PBS and appropriatelysatedilutions were platedon YPD agar medium. Plates wereincubated at 30°C and colony forming units (CFU) were counted after 1-2 days. Final CFUs per ml were determined by
    38. multiplying CFUs with dilution factor and fold-replication was determined by dividing the CFUs obtained at 24 h time-point by 2 h CFUs
    39. Single infection assay
    40. THP-1 monocytes were treated with phorbol myrsitylacetate (PMA) to differentiate them to macrophages(Tsuchiya et al., 1982). For PMA treatment, THP-1 cells grown upto 70-80% confluencewere harvested from the culture dishes at 1,000 rpm for 3 min. Harvested THP-1 cells were resuspended in 5-10 ml fresh and prewarmed complete RPMI medium. 100μlof thiscell suspensionwasappropriatelydilutedinPBS and numberof viable cells was determined by trypan blue stainingusing hemocytometer. Cell suspension was diluted with prewarmed RPMI medium to a final density of 106cells/ml. PMA was added to this THP-1 cell suspension to a final concentration of 16 nM and mixedwell.PMA-treated THP-1 cellswere seeded either in 24-well cell culture plate or culture dishes and transferred to the incubator set at 37°C and 5%CO2.After 12 hincubation, medium was replaced with fresh prewarmed medium and cells wereallowed to recover for 12 h
    41. Treatmentof THP-1 monocytic cells with phorbol myrsityl acetate
    42. weretransferred toa sterile 100 mm cell culture dishcontaining 11 mlfresh and prewarmed completemedium andculturedin tissue culture incubatorat37°C and 5% CO2.After 12hincubation, medium was replacedwith fresh prewarmed mediumand cells were allowed to proliferate till they acquire 80% confluence
    43. Freezer stocks of THP-1 and Lec-2 cells were prepared either in commercial cell preservation medium (Gibco) or completemedium supplemented with 10%heat inactivated serum and 10% DMSO. For cryopreservation, 5-6 million cells were resuspended in 0.5 mlfreezing medium in 2 ml cryopreservation vials,stored in an isopropanol bath and were transferred to-70°C freezer. Aftertwo days, freezer stocks were transferred to liquid nitrogen containertill further use. To revive the cells, freezer stocks were taken outfrom liquid nitrogen container and transferred immediately to37°C water bath. After2-3 min, when freezing medium hadthawed completely,cells
    44. Cryopreservationand revival of cell lines
    45. To isolate primary peritoneal macrophages, 6-8 week old BALB/c mice were injected with 3% (w/v) thioglycollate broth (0.55% dextrose, 0.05% sodium thioglycollate, 0.5% sodium chloride, 0.05% agar)intraperitonealy (I.P. 50 μl/g body weight). After five days of injection, mice were euthanized by CO2inhalationand peritoneal macrophages were harvested byflushing the peritoneal cavity (lavage) with 10 mlDMEM medium(Zhang et al., 2008)
    46. Isolation of primary (peritoneal) macrophages from BALB/c mice
    47. THP-1 andLec-2 cell lines were obtained from ATCC (American Type Culture Collection). THP-1 and Lec-2 cells were cultured and maintained in RPMI-1640 and α-MEM media,respectively, supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine and antibiotics (100units/ml of penicillin and 100μg/ml of streptomycin). Both cell lines were maintained at 37°C and 5% CO2in Thermo-Scientific cell culture incubator. After every 2-3 days, spent medium was replaced with fresh,pre-warmed medium. For splitting the culture, cells were harvested at 1,000 rpm for 3 min. Spent medium was discarded and cells were resuspended in 4-6 ml fresh prewarmed medium. Finally, 3-4 million cells were resuspended in 12mlmedium in 100 mm culture dishes.Cellswere cultured and maintained in tissue culture incubatorat37°C and 5% CO2
    48. Cell lines andculture conditions
    49. Animal cell culture methods
    1. The immunoblots were quantified by densitometry software ImageJ 1.17 developed by Wayne Rasband, NIH Bethesda, MD (http://rsb.info.nih.gov/nih-image).All experiments were done at least in triplicates and results were expressed as mean ±s.e.m. A two tailedStudent’s t-test was done in Graph pad to arrive at p values and differences were considered statistically significant when p-value was less than 0.05 (*p≤ 0.05), highly significant(**p≤ 0.01)andextremely significant (***p≤ 0.001)
    2. Quantification of blots and statistical analysis
    3. competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate that has been incubated for 16-20 hours at 37 °C and inoculated into 3 mlLB medium and incubated overnight at 37 °C temperature with 200 rpm shaking. 1% of this pre-inoculum was sub cultured in 100 ml LB-broth and incubated at 18 °C until OD 600 reached 0.5 -0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking. Cells were pelleted by centrifugation at 2000xg/4°C/8 min. Pellet was resuspended in 40 ml of ice-cold Innoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 xg/4°C/8 min. Pellet was resuspended in 8 ml of TB buffer inwhich final concentration of DMSO was 7% and left on ice for 10 min. 100μl aliquots were made and snap frozenin liquid nitrogen and stored at -80 °C
    4. All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl,55 mM MnCl2. 2H2O) except MnCl2were dissolved in water and pH was adjusted to 6.7 with 1N KOH. MnCl2was dissolved separately in water. MnCl2was added drop wise while stirring (MnCl2if added directly will give a brown colour to the solution and precipitates;hence it needs to be dissolved separately). Solution was then sterilized by filteringand stored. To prepare
    5. PreparationofUltra competent cells
    6. Of theligation mixture,2μl (of total volume of 10 μl reaction)was added to atube of 100μlultra competent DH5α bacterial cells and incubated in ice for 30 minutes. The tubewas quickly transferred to a water bath maintained at 42°C to give a heat shock for 90 seconds and again quickly transferred to ice. 1ml of LB broth was added to the tube and then incubated at 37°C for 1 hour. The bacterial cells were then pelletdownby centrifugation at 6000 rpm for 5 minutes and plated on LB agarcontaining appropriate antibiotic
    7. Transformation of ligated DNA
    8. A total of 100-200ng of DNA was used in each ligation reaction. Vector to insert ratio of 1:3 to 1:5was maintained. The reaction volume was generally maintained at 10μl containing 1μl of 10X ligation buffer (provided by the manufacturer) and 0.05 Weissunit of T4-DNA ligase. The reaction was carried outat 16ºC for 14-to 16-hrs or at room temperature for 4hours
    9. Ligation of DNA
    10. Digested DNA fragments required for ligation were eluted from the agarose gel after electrophoresis. The gel was visualised over a UV illuminator and section of it containing the desired DNA fragment was carefully sliced out from the gel. The sliced agarose gel was then processed using commercially available gel elution kitsfor this purpose. The elution efficiency was checked by running a small aliquot of DNA sample on agarose gel
    11. Purification of DNA by gel elution
    12. For restriction digestion(either single or double), 0.5 to 1μg of DNA was used in a reaction containing2 to 5units of commercially available restriction enzyme(s)and 5μl of the recommended buffer (suppliedas 10X concentrationsby the vendor)in atotal reaction volume of 50μl. The reaction mixture was incubated for 2 h or overnight at 37°C. The digested DNA fragments were then visualised by ethidium bromide staining after electrophoresis on agarose gels. Commercially available DNA size markers were loaded along with the samples to ascertain or estimate the sizes of the digestedfragments
    13. Restriction enzyme digestion
    14. Overnight grown bacterial culture (3ml)was pellet down by centrifugationat4ºC for10-min at 6000 rpm. The cells were re-suspended in 200μl of Resuspension solution(solutionI). 400μl of freshly prepared Lysissolution(solution II)was then added and mixed by gently inverting the tubesfor 4-6 times and allowed to lyse for 5 min at room temperature.The complete lysis was ascertained by uniformity and clarityof the contents. Subsequently, 400μl of Neutralization solution(solution III)was added and the tubes were inverted 4-6 timesand gently for homogeneous mixing followed byincubation for 5 min on ice. After centrifuging at 12,000 rpm for 15-min, supernatant was decanted into a fresh tube, and0.7 volume of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in 100μl of TE-buffer. It was treated with RNase at a concentration of 20μg/ml by incubating at 37ºC for 1hour. It was further extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixture. After centrifugation, the clear supernatant was used for recovering the nucleic acids. The nucleic acids were precipitated with 2.5 volumesof ethanolin presence of3 M sodium acetate. In case where high purity plasmid preparations are required (for transfection to cells) the plasmid isolation was carried out with the commercially available midiprep or miniprep kits following the manufacturer’s instruction. Plasmids were observed on 1% agarose gel
    15. Isolation of plasmid DNA
    16. Recombinant DNA techniques
    17. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions
    18. DNA sequencing
    19. Agarose gels were preparedby boiling appropriate amount of agarose in TAEbuffer. After dissolution, it was cooled and then poured in a casting tray containing a comb for desired number of wells. The gel was allowed to solidify and then shifted to horizontal electrophoresis tank containing TAE buffer. The DNA samples were mixed with appropriate volumes of 6X DNA loading dye, loaded on the gel andelectrophoresedat appropriate voltage and current conditions (generally 80 V,400 mA). The gel was stained in ethidium bromide solution(1 μg/ml)for 15-min at room temperature and visualisedby fluorescence under UV-light in a UV-transilluminator
    20. Agarose Gel Electrophoresis
    21. 2μg of total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase which is a commercially available version of M-MLVRT with reduced RNase H activity and increased thermal stability.According to manufacturer’s protocol1μg of RNA,1μl oligo(dT)(500ng),1μl 10mM dNTPand nuclease freewater was added to afinal volume of 13μlin a PCR tube.Thismixture was then incubated at 65°C for 5 minutesin a thermo cyclerand then quicklytransferredtoicefor 1minute. To this 4μl of 5X first strand buffer 1μl of 0.1MDTTand1μl ofRNaseOUT (40U/μl) were added. Then contents were then mixed and 1μl (200 units/μl) of SuperScript III RT was added. Themixture was then incubated at 50°C for 60 minutesin a thermo cycler.Lastlythe reaction was stopped byincubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then usedas a template for PCR
    22. RT-PCR (Reverse Transcriptase PCR)
    23. The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by considering the OD (λ260)= 1 corresponding to50μg/ml DNA and 40 μg/ml ofRNA. The purity of nucleic acids was checked by their A260/A280 ratioconsidering 1.8 for DNA and 2.0 for RNA. These measurements were done in NanoDrop 2000 UV-Vis Spectrophotometer
    24. Quantification of nucleic acids
    25. Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed from culture dish and recommended amount of TRIzol wasadded directly on to the dish and kept at room temperature for 5 minutes for lysis of cells. The cellular homogenate was then transferred to a 1.5ml microcentrifuge tube. For each mlof TRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed of 12,000 rpm for 10 minutes. The upper aqueous phase was transferred into a fresh micro centrifuge tube and RNA was precipitated by adding 500μl of iso-propanol. The RNA pellet was obtainedby centrifugation at 12,000 rpm for 30 minutes at 4°C. The pellet was washed with 1ml of chilled 70% ethanol followed by centrifugation at 12,000 rpmfor 5minutes. The supernatant was removed and the pellet air-dried for about 5 minutes. The pellet was resuspendedin 30-50μl RNase free deionisedwater and dissolved at 55ºC followed by quantificationusingnanodrop spectrophotometerfor further use.The RNA integrity was checked by evaluating the 18S and 28S rRNA signals by running 1μl of total RNA on denaturing agarose gel stained with ethidium bromide
    26. Total RNA isolation from cultured cells
    27. Molecular techniques
    28. nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm
    29. Transient transfection of plasmid DNA in cellswas performed usingLipofectamine 2000transfection reagentaccording to manufacturer’s protocol. Briefly, 0.5 to 1million cellswere seeded in a 35mm tissue culture dish one day prior totransfection. For each 35mm dish, 4μg DNA was mixed in 250μl of Opti-MEMin one polypropylene tube. In another tube 10μl of Lipofectamine 2000 was diluted in250μl Opti-MEM and incubated at room temperature for 5 minutes. DNA and Lipofectamine 2000 were mixed together and allowed to form complexes for 30minutes at room temperature. Meanwhile, the adherent cells were washed twicewithPBS and 1ml of Opti-MEM was added. 500μl of complexes were then added to each dishcontaining cells and medium. After 6-8 hrs, the medium containing complexes wasremoved and complete medium was added and transgene expression was evaluated 24-48 hrs after transfection. Since most of the experiments
    30. Transient transfection in adherent cells
    31. Themixture is incubated in a water bath at 37⁰C for 15 min and afterwards transferred on ice and 4μl of DNA loading buffer is added. The samples were then run on a polyacrylamide gel electrophoresis which had been pre-run for 30 min. Electrophoresis was carried out at 4⁰C for 3h till the bromophenol blue migrated to 2cm above the bottom of gel. The gel was taken out and kept on Whatman filter paper sheet and covered by saran wrap followed by drying in a gel dryer at 80⁰C for 1h under suction. The dried gel was exposed to phosphoimager screen by keeping in phosphoimager cassette overnight
    32. A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic
    33. Binding reaction
    34. The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O
    35. The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo
    36. end labelling of the oligonucleotides
    37. Electrophoretic mobility shift assay
    38. Adherent cells growing either on cover slips or chamber slides were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS thrice for 5 min each and blocking was done in 2% BSA(preparedin PBScontaining 0.3% Triton-X 100) for 1h.The cells were incubatedwith primary antibody(dilutedin PBScontaining 0.3% Triton-X 100)for 2h at room temperature or overnight at 4⁰C.The cells were washed with PBS thrice for 5 min each followed by incubation withAlexa Fluor 488-or 594-conjugatedsecondary (anti-mouse/rabbit) antibodiesfor 1h. Then the cells were mounted on microscopicslides using Vectashieldmountingmediumcontaining nuclear dye DAPI. Imaging was done byeither the laser scanning confocal LSM510 or LSM 750 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscope
    39. Immunofluorescence Microscopy
    40. Equal amount of proteins were loadedon an appropriate percentageof denaturing SDS-PAGE gel. After completion ofthe run, the gel was over laid on a PVDF membranecut to the size of gel and sandwiched between filter paper sheets and kept inthe blotting cassette in the presence of transferbuffer. Finally the cassette was put in themini transblotapparatus and blotting was done for 2-3hours at a constantvoltage of 80Vat 4⁰C. For blocking the nonspecific sitesmembrane was incubated with blocking solution(5% non-fat milk solution in TBST)with gentle shaking for 1 hourat room temperature. Excess milk from the membrane was washedoff with TBST and themembrane was incubatedwith primary antibody diluted in 1XTBST for 3 hours atroom temperature or overnight at 4°C withshaking. After incubation the membrane was washedwith TBST and incubatedwithappropriate secondary antibody (conjugated with horse-radish peroxidase)diluted in5% fat free milk solution (in TBST) for 1hat room temperature.The blotwas later washed thricefor 10min eachwith TBST and processed for the detection of proteinsignal using ECL-prime chemiluminescencedetection reagent followed by detectionof signal either on X-ray filmor in a chemiluminescence detectionsystem(Proteinsimple, California, USA)
    41. Immunoblotting
    42. BCA (Bicinchoninic acid) method was used to determine the proteinconcentrationin various samples. The Cu2+ions from cupric sulphate (present inBCA reagent B) reagent arereduced to Cu+by the protein in an alkaline medium. The cuprous ion (Cu+) then combines with BCA (present in BCA reagent A) to give a purple colour whose intensity is proportional to the amount of protein present in the samples. This intensity is measuredby colorimetry at 562 nm. BCA reagent was prepared by mixing reagent A with reagent B in avolumeratio of 50:1. A standard curve was generated using increasing concentrations of BSA (2-10μg) in a 25μl reaction, in a 96 well plate. Cell lysates were also dilutedto same volume in parallel wells. 200μl of BCA reagent was then added to each well and incubated at 370C for 30 minutes. The absorbance readings were then takenin a spectrophotometer at 562 nm. Total protein was quantified by calculation of the slopes of regression lines ofabsorbanceand BSA standards
    43. Protein estimation
    44. For preparation ofcellular homogenate from adherent cell culture, the medium was first removed and cells were washed with ice cold 1X PBS. The cells were then scraped in 1X PBS and pellet down by gentle centrifugation (4000 rpm for 2 minutes) at 40C. Cell lysis buffer was then added to the cell pellets and lysis was allowed for 30 minutes on a rotor at 4⁰C. Post lysis, cellswere centrifuged at 13000 rpm for 10min at 4°C. The pellet was discarded and supernatantwascollectedas cell homogenate
    45. Extraction of total cellular protein
    46. drop wiseaddition and kept at 4⁰C for 24h. Cells were then washed with PBS and stained with DNA staining solutionat 370C in darkwith intermittent shaking. The DNA content of cells was measured by flow cytometryusing FACS-Aria (Beckton-Dickenson) at 695 ±20 nm using a 655 long pass filter.The DNA content was then analysed by FACSDivaor FlowJosoftwareto evaluate the various phases of cell cycle. The diploid 2N DNA content was referred as G1/G0 population and the 4N DNA content was referred as G2/M population. Cells with intermediary DNA content (between 2N -4N) content were considered as S phasecells and those below 2N DNA content as sub G0 cells
    47. Thecells were collected at various time points by trypsinization, washed in phosphate buffered saline (PBS, pH 7.2) and fixed in chilled 70% methanol: ethanol (1:1) solution by
    48. Cell cycle analysis
    49. Cells were seeded in replicates of five @ 3X103cells per wellinfive different 96well cell culture platesand grown in complete media. The method described earlier was slightly modified and followed (Gillies et al., 1986). After every 24h of seeding, one plate was stained with 0.2% crystal violet in 2% ethanolfor 15 minutestill 4thday i.e. 96h.One plate was stained just after the cells get attached to use as 0h time point. Excess dye was removed from the plates by washing with ample amount of water. Crystal violet dye incorporated in the cells was extracted using 0.1% SDS solution by shaking for 10 minutes on a shaker. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. The experiment was repeated at least three times and the average absorbance was plotted for each time point to generate a growth curve
    50. Cell growth Assay
    51. Table2.2: Cell types used in the present study
    52. In the present thesis, various cell types were used for which the details are provided in the Table 2.2. SiHa, HeLa, HaCaT, U2OS, SaOs , A549,HPLD andHEK-293cells were grown in Dulbecco’s modified Eagle’s medium (HyClone, Thermo Scientific, Logan, Utah, USA) supplemented with 2 mM glutamine (Gibco BRL), 100 U/ml penicillin and streptomycin (Gibco BRL, Carlsbad, CA, USA), and 10% fetal bovineserum (Gibco BRL, Carlsbad, CA, USA) under humified conditions at 37°C and 5% CO2.Cells were grown in cell culture dishes till they attained 70% confluency. For sub culturing, these were then trypsinised using 0.05% Trypsin EDTA solution and incubated for 5 minutes at 370C for cells to be detached from surface. The detached cells were then collected by gentle tapping the dish and pipetting. Trypsin was then inactivated by addition of FBS containing culture medium, transferred to a 15 ml tube and centrifuged at 1500 rpm for 2 minutes in a hanging bucket centrifuge. The cell pellet was then resuspended in complete medium and counted in Neubauercell counting chamber. Viability of the cells was checked by trypan blue exclusion method.Appropriate number of cells wasthen sub cultured in fresh cell culture dishes with culture medium as per the experimental requirements
    53. Maintenance of cell lines
    54. Cell biology methods
    1. All data were presented as mean ± standard deviation (SD)/standard error of mean (SEM) fromthree independent experiments. Statistical analysis was performed using Student's t-test and one-way ANOVA followed by a post hoc Tukey testwherever applicable. Results were analyzed and illustrated by SPSS statistical software package (SPSS for Windows,version 16). Comparisons are done within and between the test groups (i.e.,parentalcells and profilin-stable cells). Asterisk (*) symbol indicates statistical difference between parentaland profilin-stable cells, whereas Number (#) and Dollar ($) signsindicatestatistical difference within parentaland profilin-stable cells, respectively. Significance of results was determined as p ≤ 0.01 and p ≤ 0.05(*indicates p ≤0.05, **indicates p ≤0.01, *** indicates p ≤0.001 and **** indicates p ≤0.0001
    2. Statistical analysis
    3. AutoDock tools 1.5.6 (Morris et al., 2009) and PyMOL (Sanner, 1999) was used to prepareand analyze the docking simulations.Preparation of protein structures for docking:The three dimensional structure of PTEN (PDB ID: 2PBD) (Lee et al., 1999)and profilin-1 (PDB ID: 1D5R) (Ferron et al., 2007)were obtained from protein data bank, PDB (www.rcsb.org).Prior to initiating the docking simulations, all non-protein molecules were removed from the intact PTEN and only the chain P was retained. In the same way, we retained (‘chain A’) for profilin-1. All the non-protein molecules were removed usingChimera(Pettersen et al., 2014).Protein PTEN was used as receptor and profilin-1was used as ligand. Kollman united atom charges and polar hydrogen were added to the receptor protein. Receptor protein was kept rigid in docking process, assuming there is no induced conformational change upon ligand binding. In contrast to the protein, torsional flexibility was permitted for the ligands via the side-groups and backbone of protein was kept rigid.Atomic affinity and electrostatic potentials were computed for a grid boxand positioned around the approximate centre of the binding site. The grid box size was set at 58.0 X 76.0 X 58.0 Å (x, y, and z) with the spacing between grid points at 1 Å and the center at 36.253 X 82.395 X 31.728 for x, y and z coordinates, respectively. All other docking parameters remained as the Autodock default settings.Analysis of docking data:AutoDockVina (Trott and Olson, 2010) software was used for docking simulations. The resulting docked conformations were clustered into families of similar conformations, with the root mean square deviation (r.m.s.d.) clustering tolerance of 2.0 A ̊. As a rule, the lowest docking-energy conformations were included in the largest cluster. The process was repeated for each remaining conformation, until all the conformations belonged to a single cluster. A new reference conformation was defined every time a new cluster was created. Following this, the best-docked conformation is selected as the lowest energy pose in the most populated cluster, i.e., the cluster with the highest convergence out of the 100 trials. This differs from the practice of simply choosing the overall lowest energy pose. Thus, we avoid picking lower energy ranked poses belonging to sparsely populated clusters, which can be considered as ‘chance-hits’ given that the docking algorithm was unable to converge to similar poses in other independent trials
    4. Molecular docking
    5. The highly efficient Ultra competent DH5α cells for transformation were prepared by Inoue method (Inoue et al., 1990). Briefly, a single bacterial colony was picked from LB agar plate, inoculated into 10 ml LB medium and incubated overnight at 37°C temperature with 200 rpm shaking. Following day, 1% of the pre-inoculum was sub-cultured in 100 ml LB-broth and incubated at 18°C until OD600 of 0.5-0.6 was reached. Culture was then kept on ice for 10 min with constant shaking. Cells were pelleted by centrifugation at 5000 rpm at 4°C for 10 min. Pellet was resuspended in 40 ml of ice-cold Inoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000g at 4°C for 10 min. Pellet was resuspended in 8 ml of TB bufferin which 560 μl DMSO was added and left on ice for 10 min. Finally, 100 μl aliquots were made by snap freezing in liquid nitrogen and stored at -80°C
    6. Preparation of Ultra competent cells
    7. In a typical ligation reaction, a total of 100 ng of vectorwas usedwhereas;theconcentration of insert varies from 300 to 500 ng. For10μlreaction volume, 1μlligation buffer (provided by the manufacturer) and 0.5μlof T4-DNA ligasewere added in vector-insertmixture. The reaction was maintainedat 16oC for 14-16h.After ligation, 2μl of the ligation mixture (of total volume of10 μl reaction) was added to a vial of ultra-competentDH5α bacterial cells and incubated in ice for 30 minutes. The ligation mixture was allowed toheat shock at 42oC for 90 seconds followed by quick transfer onice. About1ml of LB broth was added to the tube and incubated at 37oC for 1 hour. The bacterial cells were then pelleted by centrifugation at 6000rpm for 5 min and plated on LB plate containing appropriate antibiotic
    8. Ligationand transformationof DNA
    9. For construction of desired clones, 1-2 μg of DNA was used for restriction digestion. In a typical reaction, 2-5units of restrictionenzymeswereused in the total reaction volume of 50μl along with5μl ofrecommended buffer (supplied as 10X digestion buffer). The reaction mixture was incubated for 3-5 hat 37oC. The digested DNAwasthenloaded along with DNA size marker and separated on agarose gel electrophoresis. The gel was visualized over a UV illuminator and section containing the desired DNA fragment was carefully sliced out. The sliced agarose gel was then processed using commercially available Gel Extraction kit (Qiagens) for this purpose
    10. Digestion and elution of DNA
    11. Plasmid DNA was isolated from10-100ml of bacterial culture grown overnight by using commercially available Miniprep or Midiprep kits (Invitrogen) as per manufacturers’protocol. Briefly, bacterial cells werepelleted by centrifugation at 6000 rpmfor10 min.The pelleted cells werere-suspended in 300μl-4ml of resuspensionsolution containing RNase H.About300μl-4ml of alkaline lysis solutionwas then added and mixed by gently inverting the tubesuntil clear and uniform lysatewas appeared. Subsequently, 350μl-5mlof neutralizing solutionwas added,the tubes were inverted repeatedly and gently to ensurehomogeneous mixing, followed by incubation for 5 min on ice. After centrifuging at 12,000 rpm for 15 min, supernatant was passed through commercially supplied filter columns by either gravity flow or high-speedcentrifugation. During this step, plasmid DNA binds to the column and was recovered by passing elution buffer through the column. The plasmid DNA present in the elution buffer was collected into a fresh tube and 70%(w/v)of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30 min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in desired amountof TE-buffer. Finally, the purity of plasmid wasobserved on 1% agarose gel
    12. Isolation of plasmid DNA
    13. DNA sequencing
    14. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions
    15. For preparing agarose gels,appropriate amount of agarose(0.8-2%) was dissolved by boilingin TAEbuffer, until clear slurry was formed.It wasthen poured in a casting tray containing a comb for desired number of wells. The gel was allowedto solidify andshifted to horizontal electrophoresis tank containing TAE bufferwith 1 μg/ml ethidium bromide. Appropriate volumesof 6X DNA loading dye were added in the samples and subjected to electrophoresis atconstant voltage (generallyat80 V), along with appropriate DNA ladder,untilband were resolved. The gel was visualised underUV-light in a Gel-Doc or UV-transilluminator
    16. Agarose gel electrophoresis
    17. The quantityand purity of nucleic acids weredetermined by measuring the absorbance at 260 and 280 nmusing automated NanoDrop instrument. The concentration of nucleic acids was calculated by taking 1 OD at 260nm = 50μg/ml for DNA and 40 μg/ml for RNA. The purity of nucleic acids was checked by their A260/A280 ratio
    18. Spectrophotometric estimation of nucleic acids
    19. The reaction mixture was then placed on a thermo-cycler with the required cycling conditions for amplification of the desired gene, as described in Table 2.7. The amplified products were purified by PCR purification kit (Qiagen) and either quantified by spectrometry or separatedby agarose gel electrophoresis.Table2.7:Cycling conditions for PCR
    20. The PCR amplification of desired genes werecarried out using Taq polymerase reaction kit obtained from Fermentas.Typically, a reaction mixture containing primers with plasmid containing geneof interest or c-DNA was prepared as described in Table 2.6. Table2.6: Various components of PCR reaction mix
    21. Polymerase Chain Reaction (PCR)
    22. Total RNA, isolated by TRIzol method was reverse transcribed into cDNA by One step Access RT-PCR kit (Promega, Madison, WI), as per manufacturer’sprotocol.Briefly, 1 μg of RNA, 1 μloligo-dT(500ng), 1 μl 10 mMdNTP and nuclease free water was added in a PCR tube. This reaction mixture was incubated at 65°C for 5 minutes in a thermo cyclerand then quickly transferred to ice. To the mixture, 4μl 5X firststrand buffer, 1μl 0.1M DTT,1μlRNase-OUT (40U/μl) and 1 μl (200 units/μl) of SuperScriptIII were added.The contents were mixed by gentle vortexing and incubated at 37°C for 60 minutes in a thermo cycler. Finally,the reaction was stopped by increasing the temperature to72°C for 10minuteson thermo cycler. The cDNA thus prepared, wasused as a template for PCR.PCR was then performed for either amplification of the gene of interest or relative expression of desired genes by the using gene specific primers(Table 2.5). Products were separated by agarose gel electrophoresis (2%) and visualized by ethidium bromide staining.Table2.5: List of RT-PCR primersused in the study
    23. Reverse transcriptase (RT)-PCR
    24. Total RNA was isolated from the treated cells usingTRIzol method, essentially described by Donald and his co-worker (Donald et al., 2010).TRIzol is a single-phase solution of guanidinium isothiocyanate and phenolthat can concomitantly denature proteins and other biological material. Addition of chloroform to this leads to phase separation: proteins remains in organic phase whereas, DNA and RNA resolves to interphase and aqueous phase, respectively. Before starting the experiment, area was sanitized with RNAZap toremove any contamination of DNases. After treatment, culture media was gently removed from the dish without disturbing the cell monolayer. TRIzolreagentwas added directly on to the dish andcellswere allowed tosuspend in it by repeated pipetting. The cellular homogenate was then transferred into amicrofuge tubes. For each ml of TRIzolused, 200μl of chloroform was added andvortexedfor about 30 seconds, followed bycentrifugation at maximum speed of 13,000 rpm for 10 minutes. The upper aqueous phase wastransferred into a fresh micro-centrifuge tube and 500μl of ice-cold iso-propanol was addedto precipitate RNA.The RNA was pelleted by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was decanted and the pelletwas allowed towash with 1ml of ice-cold 70% ethanol followed by centrifugation at maximum speed for 10 minutes. Finally, the supernatant was removed and the pellet was allowed toair-dryfor about 5-10 minutes and solubilized in 50μl RNase free deionized (DEPC-treated Milli-Q) water and quantified by spectrophotometry for further use
    25. Isolationof total RNAfrom cultured cells
    26. Table 2.4: List of double-stranded oligonucleotides used in the present study
    27. The DNA-protein complexes were then separated from free oligonucleotides on 6.6% native PAGE gel. The samples were loaded into a native PAGE gel, which was pre-run at constant current (40-50 mA) for 15-30 minutes. Electrophoresis was performed at constant current (80-100 mA), till the bromophenol blue dye front reached 1-2 cm from bottom of the gel. The glass plate was carefully removed without disturbing the gel and the Whatmann filter paper no. 3, cut to the size of the gel, and wasplaced over it. The paper was pressed gently and the gel, which was now firmly stuck on the paper, was covered with saran wrap and kept for vacuum drying on the gel-dryer at 80oC for 1 h. After drying, gel was exposed on a Phospho-imager screen for 12-24 h and scanned on Phospho-imager to detect the band of interest.To determine the specificity of the transcription factor binding or sub-unit interacting to the desired oligonucleotide, super-shift assay was performed. For this, 8-10 μg of nuclear extracts were first incubated with desired antibodies (concentration varies for different) or their isotype control for 1h at 25°C, followed by incubation with binding reaction mixture. The various oligonucleotide sequences used in the present study are listed in Table2.4below
    28. For detection of protein-nucleic acid interaction, an electrophoretic mobility shift assay (EMSA) was conducted as described by Hellman and Fried (2007). Breifly, 8-10 μg of nuclear extract protein was incubated with binding reaction-mixture containing either 32P end-labeled double-stranded oligonucleotides (NF-κB, AP-1, p53 or SP-1) or unlabeled oligonucleotide as shown in table 2.3. The reaction mixture was incubated at 37°C for 45-60 min. After completion of reaction, 6μl of 6X DNA-loading dye was added and mixed well by gentle tapping.Table 2.3: Binding conditions for DNA-protein complexes in EMSA
    29. Gel shift assay
    30. The mixture was then incubated at 37oC for 45 min to radiolabel the oligonucleotide. Simultaneously, the Sephadex G-50 column was prepared in 1 ml syringe. The reaction mixture was loaded on the column and the eluting fractions were collected in the microfuge tube by loading 200 μl milli-Q water on the top of the column. After collecting 5-6 fractions, the tubes were analysed using a GM counter for the amount of radioactivity. The fractions having specific activity between 3.5-4.5 X 106cpm/pmoles were pooled. To this, 100 pM of the complimentary strand of the oligonucleotide was added and heated at 95°C for 5 minutes. The mixture was allowed to annealat room temperature for 1 h and further used in Gel shiftassay
    31. The oligonucleotides of different transcription factors such as NF-B, AP-1, p53 and SP-1 were 5′-end labelled using radioactive γ32-ATP (obtained from BRIT, BARC, Mumbai, India) and Polynucleotide kinase, as per manufacturers’ protocol. The reaction mixture containing the different components, described in the table 2.2below was added in a microfuge tube.Table 2.2: Various components of oligonucleotide labeling reaction mixture
    32. Radioactive labelling of oligonucleotides
    33. and stained with ‘Live & Dead’ cell assay reagent (5 μM ethidium homodimer, 5 μM calcein-AM) for 30 min at room temperature. Red (as dead) and green (as live) cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).For flow cytometry, cells were transiently transfected with either empty vector or various constructs. After 12 h, cells were treated with a combination of CHX (cycloheximide, 25 μg/ml) and TNF (5 nM) for 24 h. Cells were washed, trypsinised and then subjectedto flow cytometry (FACS Aria, BD Biosciences) using Live-Dead Cytotoxicity assay kit (Invitrogen). Live versus dead cells were analysed using FlowJo software
    34. Cytotoxicity assay: The drug-induced cytotoxicity was measured by the 3-(4,5-Dimethylthiazolyl-2)-2,3-diphenyltetrazoliumbromide (MTT) assay as essentially described by Mosmann et al., 1983. Briefly, 5x104cells/well were seeded in 96-well plate. After 12 h, cells were treated in triplicates with different agents for different concentrations and time (in a final volume of 100μl). After completion of treatment, 25 μl of MTT solution (5 mg/ml in PBS) was then added and incubated for 2 h. The cytotoxicity was evaluated by uptake and cleavage of yellow MTT dye to purple formazan crystals by dehydrogenase activity in mitochondria of the living cells. Thereafter, 100 μl of extraction buffer (20% SDS in 50% dimethlylformamide) was added. After an overnight incubation at 37ºC, the absorbance at 570 nm was measured using 96-well multiscanner autoreader(Bio-Rad) with the extraction buffer as blank. Absorbance values were normalized to untreated cells and represented in percent cell viability for different concentrations or treatments.Determination of nuclear fragmentation: The morphology of live and dead cells was observed by staining the nucleus with DNA intercalating dye, propidium iodide (PI).Briefly, cells were treated with several apoptotic inducers for different concentration or time. Thereafter, cells werewashed with PBSand fixed in ice-cold 80% methanol for overnight at 4°C. Following day,cells were washed and suspended in 100 μl of PI solution (0.1% Triton, 0.2 mg/ml RNase A and 50 μg/ml PI in PBS) for 30 min in dark. Cells were then mounted on slides and viewed under fluorescence microscope (in 560 nm filter) to determine morphology of intact or fragmentednucleus.Live &dead assay: The cytotoxicity of various drugs was determined using the commercially available Live/Dead assay kit (Molecular Probes, Eugene, OR). Live cells have intact membraneand active cellular metabolism,which allow Calcien-AM to permeate inside and get cleaved into green fluorescent compound, Calcein (ex/em ~495 nm/~515 nm) due to intrinsic cellular esterase activity. On the other hand, Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). Hence, the cell viability can be assayed by either flow cytometry or fluorescence microscopy.For imaging, cells with different drugs treatments were washed with PBS
    35. Assays for Apoptosis
    36. Secretory alkaline phosphatase (SEAP) assay: For SEAP assay, the culture supernatant was analyzed for SEAP activity essentially as per the Clontech kit protocol (Palo Alto, CA). Briefly, cells were transiently co-transfected with Lipofectamine 2000 transfection reagent, 0.5 μg of required plasmid DNA(s) with the protein of interest or empty vector, 0.5 μg of reporter plasmid containing NF-κB binding site cloned upstream of heat-stable SEAP (designated asNF-κB-SEAP)and 0.5 μg of green fluorescence protein (GFP) expression plasmid (Clonetech) in Opti-MEM media.After 6 h of transfection, cells were washed and cultured for 12 h in complete media, followed by treatment with different inducers. GFP positive cells were then counted to ensure similar transfection efficiency. At the end of treatment, cell culture-conditioned medium was harvested and 25 μl of medium was mixed with 20 μl of 5X buffer (0.5 M Tris, pH 9 and 0.5% bovine serum albumin) in a total volume of 100 μl in a 96-well plate followed by incubation at 65°C for 30 min. The plate was chilled on ice for 2 min and 50 μl of 1 mM 4-methylumbelliferylphosphate (MUP, substrate) was added to each well and incubated at 37 °C for 2 h. The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan, Lab Systems, MA) with excitation set at 360 nm and emission at 460 nm. The average number (± SD) of relative fluorescent light units for each transfection was then determined and reported as fold activation with respect to empty SEAP-transfected cells.Luciferase (Luc) assay:The cell pellet was lysed and extract was analysed as per Promega kit protocol.Briefly, cells wereco-transfected with Lipofectamine with 0.5 μg of reporter plasmid containing p53 binding site cloned upstream of luciferase (designated as p53-luciferase) and 0.5 μg of GFP constructs. After 6 h of transfection, cells were washed and cultured for 12 h, followed by treatment with different inducers of apoptosis. GFP positive cells were then counted.Cellswere pelleted down and lysed using the lysis buffer. The samples were freeze-thawed twice by storing them at -70oC to ensure total lysis. The supernatant,obtained by centrifuging the same at 11,000 rpm for 2 min was transferred to a fresh tube. About 100 μl of the substrate (Firefly luciferin, Promega) was added to the supernatant and light emission wasmeasured in luminometer by using a delay time of 2 sec andread time of 10 sec.The values were calculated as fold of activation over vector-transfected value
    37. Reporter gene transcription assays
    38. Uniquitination assay was performed as described by Choo and Zhang, 2009. Ubiquitination is an enzymatic process of the covalent attachment of polypeptide ubiquitin on specific lysine residuesof protein, which is thendegraded by proteasome complex. MG132 (carbobenzoxy-Leu-Leu-Leucinal), a proteolytic activity inhibitor of proteasome complex, is widely used to assess the stability of protein in vivo. Briefly, parental and profilin-stable cells were treated with 10 μM MG132 for 6 h. The whole cell extracts prepared in NTEN lysis buffer were then subjected to immunoprecipitation with anti-ubiquitin antibody. The analysis of ubiquitination was performed by immunoblotting with anti-PTEN antibody
    39. invivo Ubiquitination assay
    40. Retroviral based system was used for silencing of PTEN. BOSC23 packaging cells were grown in 100 mm culture dishes upto 80-85% confluency. Retroviral RNA vector containing either scrambled control shRNA or pool of PTEN shRNA along with a PCL-Ampho helper plasmid were co-transfected using Lipofectamine 2000 reagent to generate viral particles. After 48 h, supernatant containing viral particles were used to infect MDA-MB-231 cells in the presence of polybrene (8g/ml). For p53 gene knockdown, TP53 mission shRNAs obtained from Sigma Aldrich (St Louis, MO, USA) were transfected using Lipofectamine 2000 (Invitrogen, USA) and non-targeting shRNAs (Sigma) were used as controls. The cellular homogenates were prepared 36-48 h post transfection and were subjected to immunoblotting to check the levels of protein knockdown
    41. RNA interference
    42. CHX pulse chase assay was performed as essentially described by Zhou (2004). Cycloheximide (CHX), a protein biosynthesis inhibitor was used to determine the half-life and stability of protein of interest. CHX blocks translation elongation step, thereby halting the synthesis of new proteins and therefore, time course degradation of protein can be studied. Briefly, parental and profilin-stable cells were seeded in 35 mm culture dishes and treated with CHX (50 μg/ml) the following day. Cells were harvested at different time points and level of protein was determined by immunoblotting
    43. Cycloheximide (CHX) chase assay
    44. Immunofluorescence assay was carried out as described by Bhattacharyyaet al., 2010.Adherent cells weregrown either on cover slips. After treatment, cells were fixed with 3.7% paraformaldehyde solution in PBS for 15 min and permeabilised with 0.5% Triton X-100 at room temperature for 10 min, followed by blockingin PBS containing 2% BSA for 1h. Post blocking, cells were incubated with a primary antibody in PBS (1:200 to 1:500) for 2 h. After washing, cells were incubated with fluorescent-conjugated secondary antibodyin PBS(Alexa Fluor 488 or 594 goat anti-rabbit or antii-mouse, 1:1000) for 30 min. After final wash with PBS, nuclei were counterstained with DAPI containing mounting medium (Vectashield, USA). All the steps were performed at room temperature, unless otherwise stated. Images were obtained using either the laser scanning confocal LSM510 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscopes
    45. Immunofluorescence
    46. Co-Immunoprecipitation assays were performed essentially as described by Lee (Lee, 2007). For a typical immunoprecipitation assay, cells were washed with ice-cold PBS and scapped in ice-cold microfuge tube. Then, cells were lysed with NETN buffer (containing 1 μg/ml each of leupeptin, aprotinin, 10mMeach ofNaF and phenylmethylsulfonyl fluoride (PMSF))on shaking rotator in cold room for 30 min. After centrifugation, the whole cell lysate (500 μg-1 mg) obtained was incubated with 1 μg of antibody of interest(orwithisotype control)on shaking rotator in cold room for 3 h, followed by addition of 10-20 μl of ProteinSepharose A/G beads (Santa Cruz) for 1 h. The immuno-complexes bound to beads were then pelleted atlow speed centrifugation (2500 rpm for 3 min) and washed three times with NETN buffer. The proteins bound to beadswere resolved by SDS-PAGE and immunoblotting was performed accordingto standard protocol described earlie

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    47. Co-Immunoprecipitation (Co-IP)
    48. in 5% fat free milk solution in TBST (1:7000) for 45 min at room temperature and then washed thrice.The detection of signal was performed with ECL detection reagent (Amersham Biosciences) followed by detection of signal either on X-ray film (Hyperfilm-ECL, Amersham Biosciences)or in a chemidoc system (Proteinsimple, California, USA).The blot was reprobed with anti-tubulinor anti-GAPDHantibody to ensure equal loading of extracted protein
    49. Materials and Methods472.2.7 Estimationof protein concentration in cellular lysatesBradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD2.2.8 Immunoblotting(Western Blotting)Immunoblotting was performed as essentailly described by Lee (Lee, 2007). Equal amounts of protein were resolved on a denaturating SDS-polyacrylamide gel (8-12%). After completion of the run, the gel was transferred onto PVDF membrane and placed in the blotting cassette. The cassette wasthenput intothe mini transblot apparatus and transfer was done for 2-3 hours at a constant voltage of 80 V, depending on the size of the protein. Post transfer, membrane was rinsed in TBS containing 0.1% Tween-20 (TBST) and blocked with 5% non-fat milk in TBST for 1 h at 37ºC,on a gentle shaking rotator. After blocking, membrane wasrinsed thrice in TBST and incubated with primary antibodydiluted in TBST (ranging from 1:1000 to 1:10000, depending upon antibody used) for either3h at room temperature or overnight in the cold room.The membrane was then washed thrice with TBST and incubated withhorseradish peroxidase(HRP)-conjugated secondary antibody diluted
    50. Immunoblotting(Western Blotting)
    51. Bradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD
    52. Estimationof protein concentration in cellular lysates
    53. Materials and Methods462.2.5 Cell proliferation assayThe method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve.2.2.6 Preparation of whole cell, cytoplasmic and nuclear lysatesIn order to extract the total cell homogenate, the culture media was removed and cells were washed with ice cold PBS. The cells were then gently scrapped and pelleted by centrifugation at 3000 rpm for 3 minutes at 4°C. The cell extraction buffer was added to the cell pellet and placed on rotor kept in cold room for 30 minutes for cell lysis. Lysed cellswere then centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected as cellular lysate. Protein concentration was estimated using Bradfordassay, described below.For a typical Western blot assay, 30-70μg of protein was loaded on theSDS-PAGE.For the preparation of cytoplasmic lysate,ice-cold hypotonic cytoplasmic extract buffer was added in the cell pellet andgently mixed with the pipette in a microfuge tube. The cell suspension was incubated on ice for 30 minto allow them to swell.After incubation, freshly prepared 10% NP-40was added andvortexed vigorously for 15 seconds torupture the plasma membrane. The contents were then centrifuged at 13000 rpmfor a minuteat 4°C and supernatant containing the cytoplasmic lysate was transferred to another pre-chilled microfuge tube and stored at –70°C.The pellet was then further processed for extraction of nuclear lysate.For this, ice-cold nuclear extractbuffer was added to the pellet and incubated on ice for 45 min with intermittent vortexingafter every 10 min of incubation. Finally, cell suspension wascentrifuged for 5 min at 14000rpm. The supernatant containing nuclear lysatewas stored at –70°C for further experiment
    54. Preparation of whole cell, cytoplasmic and nuclear lysates
    55. The method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve
    56. Cell proliferation assay
    57. Wound healing assay(Liang et al., 2007) was employed to study the difference in migration rates ofprofilin-stable compared to parentalcells. Briefly, cells were cultured upto 90-95% confluent monolayer and a scratch was created through the cell monolayer with sterile needle. Cell debris was then removed by washing with PBS before adding the media. Images of the open gap created by this “wound” were then captured at three random locations immediately (0 h) and then at the same locations after regular interval using phase contrast microscopy untilthey are closed by migrating cells. Captured images were then used to quantify wound closure by the percentage change in the wound area per unit time and averaged for three locations for each experimental condition. During the course of the experiment, cells were maintained in 0.1% FBS containing DMEM media to ensure that wound closurewas due to the migration of cells rather thandivisonof cells
    58. Wound healing assay
    59. Briefly, cells were cultured on 35 mm culture dishes (Corning) and were transfected with pcDNA3.1 (+) empty vector or pcDNA3.1 (+) profilin-1 clone (encodes for neomycin resistance for selection in mammalian cells) using Lipofectamine 2000 reagent. The following day, cells were trypsinized and plated in four 10 cm culture dishes separately for each cell clones in selective media containing 800μg/ml of Geneticin, G418 (whose concentration was obtained from kill curve based on MTT assay). Over the time,it will select only those cells that have stably incorporated the plasmid into their genomic DNA. Cells were selected in G418 for about two weeks until colonies appeared. Meanwhile, cells were regularly washed with sterile PBSto remove dead cells from culture dishes. Colonies were picked using a pipettewith a sterile tip by loweringitto the surface of the colony of interest, followed by gentle scraping and rapid sucking.About 20 colonies were picked, followed by dilution plating in 96-well plates until single cell per well was obtained to establish a pure colony. Once colonies have reached to fair confluency, they were further expanded by dilution plating and screened for stable expression of profilinby immunoblotting and semi-quantitative RT-PCR. Once the clonal cell line is established, aliquots were freezed for future use and rest were maintained in lower concentration of G418 with proper track of passage number
    60. Generation of profilin stable MDA-MB-231 cells
    61. Transfection of plasmid DNAin cellswas performed using Lipofectamine 2000 reagent as per manufacturer’s protocolprovided with the reagent. Briefly, 0.5 to 1 million cells were seeded in a 60mm or 100mm tissue culture dish. After 12h of seeding,transfections were performed. 6-12 μg DNA was mixed in 500-1500 μl of Opti-MEM in one polypropylene tube and simultaneously, 15-30 μl of Lipofectamine 2000 was mixed in similar volumes of Opti-MEM in another tube and incubated at room temperature for 10minutes. Opti-MEM containingDNA and Lipofectamine 2000 were then mixed and incubated for 30 minutes at room temperature for the formation of DNA-lipid complex. Meanwhile, the cells were washed with sterile PBS and 4-10ml of Opti-MEM was addedin the plate. DNA-lipid complexes were then added to each dish for 6h. After that, the medium containing complexes was removed and complete medium (DMEM containing FBS) was added. Expression of transgene was evaluated 24-48hafter transfectioneitherby immunoblottingor immunofluorescence or by RT-PCR followed by PCR
    62. Transient transfection in adherent cells
    63. Maintenance of cell lines
    64. Inpresent thesis, various cell lines have been used as mentionedearlier. Cells were either cultured in DMEM or RPMI medium containing 10% fetal bovine serum (FBS)along with antibiotics such as penicillin (100 U/ml), and streptomycin (100 μg/ml).In general, cells were grownin tissue culture T-75 flaskupto 85-90% confluency. Cells are washedwith PBS, followed by trypsinization with 0.05% Trypsin EDTA solution. Cells were detachedfrom the surfaceeither by gentle tapping or gentlepipettingor incubated for 5 minutes at 37°C. Culture medium containing serum was then added to inactivate trypsin. After careful mixing, cells were transferred to a 15 ml tube and centrifuged at 800 rpm for 5minutes. The cell pellet wasre-suspended in a fresh culture media containing FBS. The cell viability was checked by trypan blue staining, followed bycounting in Neubauer cell-counting chamber. Appropriate number of cells wasthen either sub-culturedin the ratio of 1:4 to 1:6or seeded in culture dishes as per the experimental requirements.Cells were maintained in humidified incubator at 37ºC in 5% CO2-95% air, throughout the experiment
    1. For each strain calls from a single colony, picked with a microtip from the appropriate medium were suspended in 10 μL zymolyase cocktail and incubated at 37ºC for 90 min. 2 μL of zymolyase-treated cell suspension was used as the template in a 25 μL PCR reaction
    2. was fused to the 5’ region of the nat1gene. The 3’ region of the nat1gene was in turn fused to the 5’ region of UBC11, the gene that is downstream of RPA43in the genome. These two fragments were transformed into the NOY222 rpa34strain using yeast transformation kit (Clontech) and selected on nourseothricin(NAT) 200 μg/mL to identify strains carrying mutant RPA43generated by homologous recombination (Fig. 2.4) containing YPD. Colony PCR was performed using primers that amplify the merged region of RPA43fragmentand natgene to verify the site of insertion
    3. Yeast colony PCR
    4. To conduct yeast transformation competent cells were made from relevant haploid yeast strain using Frozen EZ Yeast Transformation II Kit from Zymo Research Corporation, according to the manufacturer’s instructions.To conduct plasmid shuffling in the NOY222, NOY222rpa34Δ and NOY222rpa34ΔRPA43S/322/323/325/A strainsto generate native and S/A mutant versions of the RPA190gene, the competent cells were transformed with either pRS314RPA190 or pRS314 RPA190S1413/1415/1417/A harbouring wild type a mutant versions of RPA190 generespectively,and transformants were selected by growing them on yeast nitrogen base containing leucine, G418 (200 g/mL) and canavanine (6 g/mL) but lacking uracil. The resulting strains have chromosomal deletions of RPA34 and RPA190 and harbour either wild type or mutant versions of RPA190on pRS314 plasmids
    5. genomic mutation on the A43 protein coding gene RPA43was inserted in the wild type BY4741 and NOY222 rpa34strains. Plasmid pGP5 RPA43, gifted by Dr.Herbert Tschochner, and a plasmid harbouring the nourseothricin N-acetyltransferase (nat1) gene(Goldstein and McCusker, 1999)were used for a PCR based site directedmutagenesis to create a DNA fragment to generate a genomic mutant of RPA43 (Fig

      A

    6. with 0.01% NP 40 in water, andwere diluted serially and plated on rich medium (YPD). RPA34in the genome was replaced with kanMx4that provides resistance toG418 and RPA190gene deletion was done by replacing it with URA3gene that supports growth of the strain in the absence of uracil in the medium. pNOY20, a shuffle plasmid harbours a LEU gene which supports yeast growth in the absence of uracil. Therefore germinated spores were streaked on a selection mediumlacking leucine and uracil and containing 200 g/mLG418to select the strain containing double mutation. The genotype of the strain was confirmed by growing the straindifferent combinations of media such as SC-Ura, SC-Leu, SC-Trp, SC-Met and SC-His. The procedure to generate RPA190 genome deletion in the background of rpa34Δ(NOY222 rpa34) was conducted by a colleague, Ms. Sitalakshmi Thampatty, in thelaboratory
    7. TheS. cerevisiaestrains used were BY4741 rpa34and NOY222 (Table 2.1) (Gerberet al., 2008).NOY222 harbours agenomic deletion of RPA190 with the RPA190encoded on pNOY20, a shuffle plasmid,as the genomic deletion is lethal. BY4741 rpa34(MATa), and NOY222(MATα)were mated by mixing these two strains and patched on the YPD agar. The resulting diploids were sporulated on yeast sporulating medium at 25°Cfor 2-3 weeks.Thespores were treated with 5U zymolyase and sonicated briefly in a bath sonicator to release the spores. The free spores were washed
    8. Generation of yeast mutants
    9. The pyrophosphorylation reaction was performed with proteins on beads in presence ofIP7 reaction buffer(Werneret al., 2010). 10X IP7 reaction buffer (Section2.1.6.7)was prepared, aliquoted andstored at 4°C. For the reaction, 30 μLof purified protein on GSH beads(1:1 beads in PBS suspension), 3.5μLof 10X buffer and 1 μCi of 5[β-32P]IP7were added, and made up to afinal volumeof 35 μL, and incubated at 37°C for 15 min. A 50 μLreaction was performed for proteins with low expression levels such as A190. To the reaction mix, 4X LDS sample buffer (Invitrogen) was added to a final concentration of 1X and incubated at 95°C for 5 min. The reaction mix was centrifuged at high speed and resolved on a 4%-12% gradient gel by Nu-PAGE(Invitrogen) using 1X MES buffer (Invitrogen). Proteins were transferred to a Hybond-P membrane (GE Lifescience) and the radiolabelled proteins were detected using a phosphorimager (Fuji Film FLA-9000). The membrane was blocked with 5% non-fat dry milk (Rockland)in 1X PBST(pH 7.4)for 2 h at room temperature followed by washes with 1X PBST at room temperature for 10 min three times.Proteins were detected by western blot using a rabbit anti-GST antibody. 1:5000 dilution of anti-GST antibody in 1X PBST containing 0.2% BSA, was added and incubated overnight at 4°C on a rotating platform. The membrane was washed in 1X PBST for 10 min three times, followed by incubation with HRP conjugated goat anti-rabbit IgG at 1:20,000 dilution in 5% non-fat dry milk (Rockland)in 1X PBST, for 1 h at room temperature. Membrane was washed with 1X PBST at room for 10 min three times.Protein bands were detected by using Super Signal West pico chemiluminiscence substrate (Perkin Elmer)
    10. IP7-mediated phosphorylation reaction
    11. After the HPLC run, vials numbered from 50 to 65 were surveyed using a Geiger counter, 4 vials with high counts were pooled into one vial and 5 μL of this 4 mL solution was measured in a liquid scintillation counter (Perkin Elmer). To the remaining solution, 800 μL of 50% ammonia solution was added to neutralise the pH and the tube was kept on ice. In a 50 mL conical tube, 45 mL of chilled water was taken, to which the neutralised IP7solution was added and kept on ice. A Sep-Pak cartridge [Waters, WAT020545] was equlibrated with 10 mL of ice cold deionized water using a 10 mL syringe. The Sep-Pak cartridge was attached to a 60 mL syringe and 50 mL of diluted IP7 solution was passed slowly through ot so that IP7would bind to the Sep-Pak column. The cartridge was washed with 8 mL of chilled water, followed by chilled 8 mL 0.2 M triethylammonium bicarbonate solution pH 8.5 (4 mL of 1.5 M triethylammonium bicarbonate, pH 8.5 + 26 mL chilled water). The bound IP7 was eluted in 4 mL of 1.5 M triethylammonium bicarbonate solution, pH 8.5, into three 1.5 mL microfuge tubes. The eluted IP7 was concentrated in a vacuum concentrator (Scanvac) at 2000 rpm, 25°C, to obtain 30-50 μL of a 1-2 μCi/μL solution of radiolabelled IP7
    12. Purification of IP7 by anion exchange cartridge
    13. of ice cold deionized water (water was kept in the cold room overnight) was added to a 250 mL volumetric flask which was placed on ice. To the water, 52.1 mL of triethylamine solution (~7.2 M, Cat. No-T088, Sigma-Aldrich) was added with the help of a glass measuring cylinder, and mixed well. The volume of the solution was made up with ice cold water till the mark on the volumetric flask, and the flask was mixed well. A pH meter (Eutech instruments 510) was calibrated using pH 7.0 and pH 10.0 solutions (Eutech instruments 510). Approximately 100 mL of 1.5 M triethylamine solution from the volumetric flask was added to a 250 mL conical flask that was placed on ice. A magenetic bead was placed in the flask and the ice bucket was placed on the magnetic stirrer. The pH measuring probe was immersed into the solution, and CO2was bubbled through the triethylamine solution and stirred until the pH reached 8.5. The conical flask was covered with paraffin film and kept on ice until the solution wasused (within 1-4 h)
    14. During the HPLC run,a triethylammonium bicarbonatesolution(1.5 M) was prepared. Triethylamine and water mixture generates heat, and therefore, approximately 100 mL
    15. Preparation triethylammonium bicarbonate solution
    16. On the HPLC system (Waters Empower Software), the instrument method was set to the programme ‘tritium small coloumn’ and the method set was set to ‘tritium small column’. The ‘set up’ icon was selected and once the flow rate was 1 mL/min, the ‘prepare’ icon was selected. A gradient was generated by mixing buffer A and bufferB as described below (Section 2.1.6.6). The injector was moved to the load position and sample was injected using a 1 mL syringe. The injector was moved to the inject position and the ‘inject’ icon was pressed immediately. On the fraction collector, the ‘run’ button was pressed immediately. The syringe was rinsed 5 times with buffer A
    17. HPLC set upA strong anion exchange partisphere SAX 4.6 mm diameter and 125 mm length column (Whatman) was fixed to the HPLC system (Waters 515 pumps). The column was equilibrated with buffer A (1 mM EDTA) (Section 2.1.6.6) overnight at 100 μL/min flow rate.Before starting the HPLC run, the fraction collector (BioRad 2110) was prepared by placing the outlet tube to vial 1 on the fraction collector. Fresh FACS tubes were placed at fraction numbers 40-65 on the fraction collector. The flow rate on the fractioncollector was set to 1 mL/min and it was kept ready.The SAX column was allowed to equilibrate (warmup programme) with Buffer A by slowly increasing theflow rate from 0.1 to 1 mL/min over a period of 70 min
    18. Purification of radiolabelled IP7by HPLC method
    19. The reaction mix was mixed well using a 200 μLpipette and 20μLeach was dispensed into eight 1.5 mL microfuge tubes. To this reaction mix 5μLof purified hexa histidine tagged IP6K1 enzyme (2-3 μg) was added. Tubes were placed in an acrylic box and incubated at 37°C overnight in a hybridization oven. The next morning, reactions were pooled into two 1.5 mL microfuge tubes containing 100 μLeach.100μLof 0.6 M perchloric acid was added to neutralise the reaction, the tubes were kept on ice for 1 min, and 33.5 μL of 1 M potassium carbonate with 5 mM EDTA was added and mixed by gentle tapping. CO2was liberated leaving a white precipitate, while tubes were kept open on ice for 1 h with gentle tapping at 15 min intervals.Tubes were centrifuged at 12000 x gfor 2-5 min and the supernatant from each tube was pooled into a new 1.5 mL microfuge tube
    20. to hold 1.5 mL microfuge tubes.Acrylic shield was used through out to block the β radiation
    21. Synthesis of 5[β-32P]IP7was conducted as described earlier (Azevedoetal., 2010). An acrylic box was placed at a 37°C in a hybridization oven before settingup the reaction
    22. Synthesis of radiolabelled 5-IP7
    23. GST fusions of yeast RNA Pol I subunits were purified as described in (Werneret al., 2010). Yeaststrainsover expressing GST tagged RNA Pol I proteins were grown overnight at 30°C in 10 mL of SC-Ura medium with 2% glucose medium. Cells were pelleted, washed in SC-Ura with galactose. Protein expression was induced by transferring the entire pellet into200 mL of SC-Ura with 2% galatose to give a final OD600of 0.8-1.0. For proteins A190 and A43 that express at very low levels, the overnight culture volume and induction volume were doubled. Cells were cultured at 30°C harvested at 3.0-5.0 OD and washedwith ice cold water. The cell pellet was suspended in 5 mL of ice cold Buffer A (Section 2.1.6.7), 750 μL ofcell suspensions were aliquoted into 1.5 mL microfuge tubes and to this 500 μL glass beads were added. Cells were lysed by bead beating using a vortex mixer (VortexGenei -2 with mix-mate attachment), and the lysate was centrifuged at high speed for 15 min at 4°C. Supernatants were dispensed into a 15 mL conical tube and Triton X-100 was added to a final concentration of 1%. Pre-swollen glutathionebeads were washed in Buffer B(Section 2.1.6.7)from which 200 μL of 1:1 bead suspension was added to approximately 5 mL of A34 and A43 expressing cell lysate and 100 μL of 1:1 bead suspension was added to 5 mL of A190 cell lysate and incubated for 2 h at 4°C on a rotary mixer. Lysates were centrifuged at 5000 xgfor 2 min and the beads were washed with ice cold Buffer C (Section 2.2.6.6) twice. Beads were further washed with ice cold Buffer B followed by ice cold 1X phosphate buffered saline (PBS) twice. Beads were suspended in an equal volume of 1X PBS with protease inhibitor cocktail (Sigma)
    24. Protein expression and purification from yeast
    25. recovery, cells were centrifuged at 2,500x gfor 4 min. The medium supernatant was discarded and cells were resuspended in 200 μLfresh sterile LB medium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 hto allow growh of individual colonies
    26. E. coli DH5α strain was transformed with yeast plasmids carrying appropriate inserts that express S.cerevisiaeproteins (Sambrook and Russell, 2001). Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 20μLligated plasmid was added to 100 μLultracompetent cells,and the cells were incubated on ice. After 30 min, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred to ice for 2-3 min. Next, 900 μLLB medium was added and cells were allowed to recover for 45 min on a shakerincubator set at 37°C. After the
    27. Bacterial transformation
    28. A single colony of E. coli DH5α strain was inoculated in 10 mL LB medium and incubated at 37°C overnight. Overnight culture was subculturedin 250 mL of LB medium of about 0.1 OD and incubated at 18°Cfor36 htillthe OD600reached approximately0.5. Cultures were centrifuged at 2,500 x gfor 10 min at 4 ̊C and harvested cells were washed gently with 80 ml ice-cold Inoue transformation buffer(Section 2.1.6.2). Cells were collected by centrifugation at 2,500x g for 10 min at 4 ̊C and gently resuspended in 20 mLice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. The cell suspension waskept on ice for 30min,and 100 μLvolume was aliquoted into pre-chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C
    29. Preparation of E. coli DH5α ultracompetent cells
    30. and A190middle (557-1100 a.a)fragments were amplified from the plasmid pRS314RPA190gifted byDr. Herbert Tschochner with primers containing BamHI and XhoI (Table 2.4).The mutant versions of A34 and A190 fragments were generated by overlap extension PCR based method. p416GPD GST-RPA43 used in ChIPwas generated by extracting the GST-RPA43fragmentsfrom pYesGex6p2 GST-RPA43by restriction digestion with HindIII and XhoIfollowed by ligation into p416GPD. Clones were verified by bacterial colony PCR, sequencing and western blot. For colony PCR a small amount of bacterial colony was taken with the help of tooth pick and touch the bottom of the 0.2 mL PCR tube to which PCR reaction mix was added and performed PCR
    31. The nomenclature system for RNA Pol I subunits, gene and protein name is given in Table 2.4 and The S.cerevisiaeRNA Pol I subunits Uaf30, A34.5, A43 full length and fragmentsof A135 (1-112 a.a) and A190 (1101-1664 a.a) were PCR amplified from genomic DNA of the wild-type strain using high fidelity DNA Taq polymerase, with primers carrying restriction sites for BamHI and NotI or BamHI and XhoI (Table 2.4). Amplified fragments were cloned downstream of the GAL4promoter in the pYesGex6p2 plasmid (Werneret al., 2010).These plasmids were generated by a colleague,Mr. Unnikannan CP,in the laboratory. Fragments and mutant versions of full length RNA Pol I subunits A34.5 and A43 weregenerated using above plasmids as templates (Table 2.3).A190 N-terminal (1-556 a.a)Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23Table. 2.6 The gene name and correspondingprotein name for RNA Pol Isubunits.RPA represents RNA polymerase I and protein name starts with A which represents that the subunit is present in RNA pol I
    32. Cloning of S.cerevisiaeORFs
    33. Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23