Mesomorph-endomorph:

Mesomorph-endomorph:

Balanced ectomorph:

Balanced mesomorph:

Balanced endomorph:

SOHATOCHART

Somatotyping Children

Limitations of the Rating Form

Third Component Rating

Second Component Rating

First Component Rating

SOMATOTYPING

Calf

Biceps

Femur

Humerus

Calf

Blood was drawn from appropriate source into heparinised tubes. The blood sample was centrifuged at 4000 rpm for 15 min ( 4 °C). The supernatant was discarded, and the erythrocytes (pellet) were subsequently washed thrice with chilled isotonic buffer [0.01 M PBS (pH 7.4)] by centrifugation at 4000 rpm and 4°C for 15 min. The washed erythrocytes were lysed in water. The resultant red cell lysate was then dialyzed extensively against PBS (pH 7.4) at 4°C to obtain stripped hemoglobin (hemoglobin devoid of bound allosteric modulators like BPG). The stripped hemoglobin was then loaded onto a pre-equilibrated DE52 column (30cm x 15cm) after extensive dialysis against 0.05 M tris acetate buffer (pH 8.5). The protein was eluted from the column employing a linear gradient of 500 ml each of 0.05 M tris acetate (pH 8.5) and 0.05 M tris acetate (pH 7) at a flow rate of 50 ml/hour. The purified hemoglobin was estimated spectrophotometrically at 540 nm (molar extinction coefficient= 53236 cm-1/M) and stored at -70°C till further use.

Purification of hemoglobin from bloo

20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7

Stock Solutions:

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

Solutions:

stored. To prepare competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate, inoculated into 3 ml LB medium, and incubated overnight at 37°C temperature with shaking at 200 rpm. 1% of this pre-inoculums was sub cultured in 100 ml LB-broth and incubated at 18°C with shaking until OD at 600nm reached 0.5 - 0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking.Cells were pelleted by centrifugation at 2000 g at 4°C for 8 min. Pellet was resuspended in 40 ml of ice-cold TB buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 g at 4°C for 8 min. Pellet was resuspended in 8 ml of TB buffer in which final concentration of DMSO was 7% and left on ice for 10 min. 100 μl aliquots were made and snap frozen in liquid nitrogen and stored at -80°C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.2H2O) except MnCl2 were dissolved in milliQ water and pH was adjusted to 6.7 with 1N KOH. MnCl2 was dissolved separately in mill Q water. MnCl2 was added drop wise while stirring (MnCl2 if added directly will give a brown color to the solution and precipitate out, hence it needs to be dissolved separately). Solution was then filter sterilized and

Ultra Competent Cells Preparation

microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots

The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml

Preparation of cell extracts

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Calcium chloride method

and the aqueous phase transferred to a fresh tube. The aqueous phase was further extracted successively, first with phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform:isoamyl alcohol (24:1). DNA was precipitated from the clear supernatant by the addition of 0.6 volumes of isopropanol. The chromosomal DNA was either spooled out or pelleted at this stage, washed with 70% ethanol, air-dried, and dissolved in suitable volume of TE buffer

The method as described in the manual Current Protocols in Molecular Biology was followed for preparation of chromosomal DNA. Cells from 1.5 ml stationary phase culture were recovered by centrifugation and resuspended in 567 μl of TE buffer. To this, 30 μl of 10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cell suspension mixed and incubated at 37°C for 1 h. Next, when the suspension looked cleared, 100 μl of 5 M NaCl was added, thoroughly mixed, followed by the addition of 80 μl of CTAB/NaCl (10% cetyltrimethylammonium bromide in 7 M NaCl) and vigorous mixing (by inverting the microfuge tube). The suspension was incubated at 65°C for 10 min, brought to room temperature, extracted with an equal volume of chloroform-isoamyl alcohol (24:1 v/v)

Extraction of chromosomal DNA from bacterial cells

high osmolarity conditions (Gowrishankar, 1989; Csonka, 1989) for β-galactosidase assay

Assays for determination of β-galactosidase enzyme activity in cultures were performed as described by Miller (1992) after permeabilizing the cells with SDS/chloroform, and the activity values were calculated in Miller units, as defined therein. For determination of proU activity from a proU::lac fusion that contains the proUpromoter cloned upstream of the lacZYA genes (as in plasmid pHYD272), cultures used were grown in LBON or K-medium (low osmolarity medium) since proU is also induced under

β-Galactosidase assay

The galEp3 (galE490∗)mutation represents a 1.3 kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to rho dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3mutant or its derivatives was monitored by one of two means. In the first, MacConkey galactose indicator plates (with 1% galactose) were used, where Gal+ colonies are red, and Gal− colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression. In the second method, growth of strains on minimal-galactose (0.2%) was used as a test for Gal+ phenotype

galEp3 assay

Lac+ colonies were distinguished from Lac− on MacConkey-lactose plates or on Xgal indicator plates. Xgal is a non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl moieties and hence, the Lac+ colonies on Xgal indicator plates are seen as dark blue colonies. Xgal was prepared as a stock solution of 5 mg/ml in dimethyl formamide and used at a final concentration of 25 μg/ml. On MacConkey-lactose medium (pH around 7.1) on the other hand, Lac+ strains can utilize the lactose sugar present in the medium to lower the pH of the medium to 6.8, resulting in a pink coloured colony while Lac─ strains are unable to utilize lactose to give a white colour

Lac phenotype

A single plaque of λ contains approximately 105-106 pfu/ml. The method of propagation of λ from a single plaque was as follows. The contents of a single isolated plaque were drawn into a 1-ml pipette tip and dispensed into 0.2 ml of LB broth. After addition of a drop of chloroform, the contents were vortexed and centrifuged. The clear supernatant was mixed with 50 μl of λ-sensitive cells and incubated for 20 min at room temperature for adsorption. 10 ml of Z-broth supplemented with 5 mM MgSO4 was then added to the infection mixture, and incubated at 37°C with shaking until lysis. The lysate thus obtained usually contained 109pfu/ml

Preparation of λ lysate by propagation from a single isolated plaque

To quantitate the P1 phage lysate preparations, titration was done using a P1-sensitive indicator strain such as MG1655. 100 μl each of serial dilutions of the phage (typically 10−5, 10−6) were mixed with 0.1 ml of the fresh culture grown in Z-broth. After 15 min adsorption at 37°C without shaking, each mixture was added in a soft agar overlay to Z agar plates, and incubated overnight at 37°C. The phage titre was calculated from the number of plaques obtained on the plates

To 0.3 ml of infection mixture, 10 ml of Z-broth was added and incubated at 37°C with slow shaking until growth followed by the visible lysis of the culture occurred (in ~ 4-6 h). The lysate was treated with 1 ml of chloroform, centrifuged and the clear lysate was stored at 4°C with chloroform

Broth method

require high fidelity,Taq DNA Polymerase from MBI Fermentas was used. However,for precise amplifications either Herculase Fusion or PfuDNA polymerasefrom Stratagene was used. Approximately, 10-20ng of plasmid or 100 to 200 ng ofchromosomal DNA was used as a template in a 50 μl reaction volume containing 200μM of each dNTP, 20 picomoleeach of forward and reverse primer and 1.5 units of DNA polymerase.In the case of colony PCR performed to examine multiple colonies for presence of the plasmid clones, E. coli cells from afreshly grown plate wereresuspended in 50 μl of sterile Milli-Q water to get a cell suspension (~109cells/ml)and 4 μl from this was usedas the source of DNA template. To verify various pMU575 clonesdescribed in this study, by colony PCR,the vector specific primer pairs JGJpMUF and JGJgalK were used. The expected amplicon for pMU575 alone is ~300-bp, while that carrying the cloned fragment would be >300-bp.For each PCR reaction, the samples were subjected to 30-cycles of amplification and the typical conditions were as follows (although there were slight alterations from one set of template/primerto another):The initial denaturation was carried out at 95°C for 4-min and the cycle conditionswere as given below:Annealing 45ºC to 50°C 1-minExtension 68°C (1-min/kb of DNA template to be amplified)Denaturation 95°C 1-minAfter 30 cycles of PCR, the final extension step was carried out again for 10-min at68°C

For amplification of short length (100-200-bp)DNA fragmentsor that do not

Polymerase chain reaction (PCR)

Overexpression and purification of ArgPand ArgPdproteins

argP+, argPd-S94L, argPd-P108S, argPd-P274Sfragment downstream of the phage T7-promoter, such that the encoded proteins beara C-terminal His6-tag provided by the vector DNA sequence. Theresultant plasmid was transformed into strain BL21(DE3) which has the T7 RNA Polymerase under the isopropyl thio-β-D-galactoside (IPTG) inducible lacUV5promoter.The resultant strains were grownin LB (500-1000 ml) to an A600of around 0.6and were then induced with 1 mM IPTG and harvested after 4-hrs of induction.Bacterial cells were recovered by centrifugation, resuspended in 20 ml of lysis buffer(20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mM DTT and 10 mM imidazole) containing20 μg/ml lysozyme, and lysed by sonication with 30-sec pulses for 10-min. Theprotocol for His6-ArgP(ArgPds)protein purification involved (i) passing the lysate through a 5ml Ni-NTA (Qiagen) chromatographic columnequilibrated with lysis buffer, (ii) washing thecolumn with 100 ml of washing buffer (20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mMDTT; 30 mM imidazole), and (iii) elution of His6-ArgP(ArgPds)from the column with elutionbuffer (20 mM Tris-Cl, pH-8;300 mM NaCl; 10 mM DTT and 250 mM imidazole) andcollection of 1.5 ml eluate fractions (10 fractions). The fractions were tested forprotein by Bradford method and the protein-carrying fractions (generally tubes 2 to 5)were pooled and dialysed in a 1:200 volume ratio against 20 mM Tris-Cl, pH-8 with 10mM DTT, 300 mMNaCl for 5 hrs followedby a change to buffer of composition 20 mM Tris-Cl, pH-8 with 10 mM DTT, 300 mM NaCl and 40% glycerol for 24 hrs. The proteins were concentrated by centrifugation toaround 1 mg/ml by using Amicon filter (pore size 10-KDa) and stored at −20ºC or −70ºC

For preparing ArgP and ArgPd-S94L, -P108S and -P274S proteins, derivatives(designated as pHYD1705, pHYD2678, pHYD2679 and pHYD2680 respectively) of the plasmidvector pET21b (Novagen) was constructed which carries the PCR-amplified

werethen recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed oncewith 70% ethanol, air-dried and re-suspended in 100 μl of TE-buffer. It was treatedwith RNase at a concentration of 20 μg/ml by incubating at 37ºC for 1-hr. It was furtherextracted with an equal volume of phenol:chloroform mixture followed bychloroform:isoamyl alcohol (24:1) mixture. After centrifugation, the clear supernatantwas used for recovering the nucleic acids. The nucleic acids were precipitated with 200μl of alcohol in presence of 0.3 M sodium acetate (Sambrook and Russell, 2001). In casewhere high purity plasmid preparations are required (DNA sequencing) the plasmidisolation was carried out with the commercially available kits following themanufacturer’s instruction. Plasmids were observed on 1% agarose gel

1.5 ml of cells from an overnight culture waspelleted by centrifuging in cold (4ºC) for10-min at 6000 rpm. The cells were re-suspended in 200 μl solution I (50 mM glucose; 25 mM Tris-Cl, pH-8; 10 mM EDTA, pH-8) with vortexing. 400 μl of freshly preparedsolution II (0.2% NaOH, 1% SDS) was added and mixed by gently inverting the tubes.Subsequently, 300 μl of solution III (prepared by mixing 60 ml of 5 M CH3COOK,11.5 ml glacial acetic acid, 28 ml water) was added and the tubes were invertedrepeatedly and gently for homogeneous mixing followed by incubation for 5-min onice. After centrifuging at 12,000 rpm for 15-min, supernatant wasdecanted into a freshtube, an equal volume of iso-propanol was added, the precipitated nucleic acids

Isolation of plasmid DNA

A differential gene expression microarray with respect to argP was performed by Genotypic Technology Pvt.Ltd., Bengaluru. The experiment was performed on an oligonucleotide microarray having 10828 probes for coding region(on average three probes were designed for each 4294 coding regions) and 4380 probes for non-coding region (on average two probes were designed for 2240 non-coding regions). The RNA was labelled using Cy3 and single channel detection was used. Data was analysed using GeneSpring GX Version 7.3

Microarray details

supplemented with amino acids and appropriate antibiotic and grown at 37ºC to an A600of 0.5-0.6. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysedwith addition of one drop of chloroform and 1-2 drops of 1% SDS solution. 0.2 ml offreshly prepared 4 mg/ml ONPG was added to start the reaction and incubated at roomtemperature till the color of the reaction mixture turned yellow. 0.5 ml of 1 M Na2CO3was added to stop the reaction and the time duration from initial addition of ONPG tothe stopping of reaction was noted.The absorbance of reaction mix was taken at 420nm and 550 nm. The A600of the culture used was also noted. The enzyme specificactivity (in Miller units) was calculated using following equation:β-galactosidase specific activity = [1000 X A420-(1.75 X A550)] / t X v X A600Where t isthe time period in minsand v the volume of culture used in ml.Each value reported is the average of at least three independent experiments, and the standard error was <10% ofthe mean in all cases

β-galactosidase assay was performed according to Miller (1992). An overnight grownculture of the bacterial strain was sub-cultured in glucose Minimal A medium

β-galactosidase assay

Thialysine or thiosine (S-Aminoethyl-L-cysteine)is a toxic analog of Lys. Strains were testedfor sensitivity/resistance to thialysine by streaking them on minimal A-glucose platessupplemented without and with100-200 μg/ml thialysine(Steffes et al., 1992)

Test for thialysine resistance

For testing ArgR+/–phenotype, the colonies werestreaked on minimal A-glucose plates containing uracil (40 μg/ml) and CAN(65 μg/ml). Uracil wasadded to the medium to sensitize an argR+strain to CAN. An argR+strain is inhibited at65 μg/ml CANon a uracil-containing plate, whereas on a plate without uracil, argR+would grow even at 700-800 μg/ml CAN. Uracil represses the carAB transcription, whichencodes the carbamoyl phosphate synthase enzyme (CarAB). This results in reducedamounts of carbamoyl phosphate, which is the common intermediate between pyrimidineand Arg biosynthetic pathways. Reduced carbamoyl phosphate levels would result indecreased flux through the Arg biosynthetic pathways. This in turn would result indecrease in Arg pools inside the cell. An argR mutant would be derepressed for the Argbiosynthetic pathway and is resistant even to 300 μg/ml CANin a uracil-containing plate

Test for ArgR+/–phenotype

Test for canavanine (CAN) sensitivity

CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil

The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison

NaCl-sensitivity testing

agar platesLac+colonies will appear dark pink colonies whereas Lac–will remain colourless

A. lacphenotype

Scoring for phenotypes

Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC

Preparation of high efficiency competent cells

For routine plasmid transformations, following method which is modification of thatdescribed by Cohen et al. (1972) was used. An overnight culture of recipient strain wassub-cultured 1:100 in fresh LB medium and grown till mid-exponential phage. Theculture was chilled on ice for 15-min, and the steps thereafter were performed at 4ºC.20 ml of culture was centrifuged and pellet was re-suspended in 10 ml of 0.1 M CaCl2.After 15-min of incubation on ice, the cells were again centrifuged and re-suspended in2 ml of 0.1 M CaCl2. The suspension was incubated on ice for 30-min. To the 200 μl aliquot of the cell suspensionplasmid DNA (20 to 200 ng in less than 10 μl volume)was added, incubated for half an hron ice and given a heat shock for 90-sec at 41ºC.The cultures was rapidly chilled, mixed with 0.8 ml of LB-broth and incubated at 37ºCfor 1-hr, and plated on an appropriate selective medium at various dilutions. An aliquotof cell suspension to which plasmid DNA was not added served as a negative control

A. Calcium chloride method

Transformation

the infection mixture was centrifuged, washed in 5 ml of citratebuffer and plated without phenotypic expression

To 2 ml of fresh overnight culture of recipient strain, 108pfu equivalent of phage lysatewas added and incubated at 37ºC without shaking for 15-min to facilitate phageadsorption. The un-adsorbed phage particles were removed by centrifugation at 4000rpm for 5-min and pellet of bacterial cells was re-suspended in 5 ml of LB-brothcontaining 20 mM sodium citrate to prevent further phage adsorption. This wasincubated for 30-min at 37ºC without shaking to allow the phenotypic expression of theantibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspendedin 0.3 ml citrate buffer. 100 μl aliquots were plated on appropriate antibioticcontaining plates supplemented with 2.5 mM sodium citrate. A control tube withoutaddition of P1 lysate was also processed in the same way. In the case of selection ofnutritional requirement,

Phage P1 transduction

0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107plaqueforming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption wasallowed to occur at 37ºC for 20-mins. To 0.3 ml of infectionmixture, 10 ml of Z-broth was added and incubated at 37ºC withslow shaking until the visible lysis of the culture occurred (in 4-6 hrs). The lysate wastreated with 0.3 ml of chloroform, centrifuged and the clear lysate was stored at 4ºCwith chloroform.Preparation of P1 lysates on recA mutant strains were also donesimilarly, but with a higher multiplicity of infection (i.e. 108starter P1 phage).To quantitate the P1 phage lysate preparation, titration was done using P1 phagesensitive indicator strainsuch as MG1655. 100 μl each of dilution of phage (typically10–5, 10–6) were mixed with 0.1 ml of fresh culture grown in Z-broth. After 15-min ofadsorption at 37ºC without shaking, each mixture was added on a soft agar overlay ofZ-agar plates and incubated overnight at 37ºC. The phage titer was calculated from thenumber of plaques obtained on the plates

Phage P1 lysate preparation by broth method

Genetic techniques

For SEM, C. glabratacells were fixed for 24 h in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) at 4 ̊C, post-fixed in 2% aqueous osmium tetroxide for 4 h and dehydrated. After drying to critical point, mounted samples were coated with a thin layer of gold for 3 min using an automated sputter coater and visualized by SEM (JEOL-JSM 5600)

Scanning electron microscopy

min. Cells were normalized to equal OD600, resuspendedin 1 ml 50 mM Tris-HCl (pH 7.5) and transferred to 2 ml microcentrifuge tubes. Cells were lysed with glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)asdescribed earlier.Brokencells were washed from glass beadswith 500 μl Tris-HCl (50 mM, pH 7.5) and pelleteddown at 15,000 g for 10 minto obtainall cell wall and membrane content. Pellet was then boiled for 10 minin 1mlTris-HCl(50mM; pH 7.5)solutioncontaining 2%SDS. SDS-extractable material(mannoproteins)was savedand remaining pellet wasboiled again in 500 μl Tris-HCl(50 mM; pH 7.5)buffer containing 2%SDS. Cell wallwas collectedby centrifugation at 15,000 g for 10 min, washed twice with1 ml waterandresuspendedin 100 μl 67 mM potassium phosphatebuffer. This washed cell wall materialwas used for β-glucan estimation as described below

Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5

Crude cell wall isolation

Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard

Crude vacuolar membrane extraction

Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells

Estimation of trehalosecontent

To assess the activity of plasma membrane proton pump, CgPma1, in cells grown in differentexternal pH environment,whole cell acidification assaywas carried out.This assay is a measurement of glucose-responsive proton pump activityin live cellsand is based on a decrease inthe pH of a weakly-buffered solutionupon extrusion of H+ions from thecell. The amount of change in the pH of the medium represents a crude measurement of the activity of functional plasma membrane proton pump in live cells. Whole cell acidification assay was conductedwithcellsgrown in YNB pH 5.5 and YNB pH 2.0medium as described previously (Martinez-Munoz and Kane, 2008) with slight modifications.After growth at30 ̊C for 2 h, cells were harvested, washed and resuspended(1.5-3.0 mg wet weight/ml) in 15ml MES/TEA (1mM; pH 5.0) buffer. Cell suspension was kept at 25 ̊C with continuousagitation. Extracellular pH of the buffer solution was recorded at 1 mininterval for 20 minwith the help of a pH meter(BT-600, BoecoGermany). To activate plasma membrane proton pumping, glucose and KCl were added to a final concentration of 40mM after 3 and 8 minincubation, respectively. Plasma membrane proton pump activitywas plotted as a change in the pH of the extracellular solutionversustime

Whole cell acidification assay

Log-phase yeast cell cultures were harvested and total protein was extracted by lysingyeast cells using glass beads. Briefly,10 mllog-phase yeast culturesgrownin appropriate medium were harvested,washed once with ice-cold water and suspended in 250 μl homogenizing buffer containing 1 mM phenylmethylsulfonylfluoride(inhibitsserine proteases), 10 mM sodium fluoride(inhibit Ser/Thr and acid phosphatases), 1 mM sodium orthovanadate (inhibits Tyr and alkaline phosphatases) and 1X concentration of protease inhibitor cocktail(RocheCat # 04693159001). Cells were lysedwith glass beads by vortexing five times at high speed for 1 min with intermittent 1 min ice breaks. Unbroken cells and cell debris were removed by centrifugation at 1,000 g for 5 min at 4 ̊C. Cell lysate was collected and protein was quantified using bicinchoninic acid (BCA)protein assay kit (Thermo Scientific # 23227) as per supplier’s instructions

Protein extraction

Themethod was used for isolation of good quality genomic DNA that wasused to map Tn7insertionin C. glabratamutants.Briefly,10 mlsaturated yeast culturewasharvested, resuspendedin 1 ml sterile water and transferred toa2 ml microcentrifuge tube. Cells were pelleteddown by centrifugation at 4,000 rpm for 5 min. Supernatant was discarded and the pellet was resuspendedin 500 μl freshly prepared solutioncontaining100mM EDTAand 5% β-mercaptoethanol andincubated at 42 ̊C for 10 min. After incubation,cells were spun down at 5,000 rpm for 1 minand resuspendedin 500μl freshly-prepared BufferB. One tip full of lyticase(Sigma # L4025) was added and cellsuspension was incubated at 37 ̊C for 1 h. Following incubation,cell suspension was spun down at 6,000 rpm to recover spheroplasts.Spheroplasts weregently resuspendedin 500μl BufferCand DNA was twice extracted with 500μl phenol:chloroform:isoamyl alcohol (25:24:1)solution.Aqueous layer was collected in a new 2ml microcentrifuge tube and DNA was precipitated with 1ml ethanol and 1/10thvolume of 3M sodium acetate (pH 5.2)by centrifugation at 13,000 rpm for 5 min. Pellet was resuspendedin 200 μl TE containing 0.3 μl of RNase Cocktail™and incubated at 37 ̊C for 30 min.After incubation, 300 μl additional TE was added and DNAwas re-precipitated withethanol and 3 M sodium acetateas described above. Pellet was washed with 70% ethanol anddried under air. DNA pellet was finally suspended in 100 μl TE and stored at -20 ̊C

Protocol III(Spheroplast lysis method

phenol:chloroform:isoamyl alcohol (25:24:1)was added to the tube and mixed thoroughly.Aqueous phase was collected after centrifugationat 12,000 rpm for 3 minand was transferred toanew 2 ml microcentrifuge tube.1 ml absoluteethanol was added to the aqueous phase and DNA was precipitated by centrifugation at 12,000 rpm for 8 minat 4 ̊C.DNA pellet was washed with chilled 70%ethanol and dried under air. DNA pellet was resuspendedin 50 μl TE containing 0.3 μl of RNase Cocktail™(Ambion®# AM2286)and incubated at 50 ̊C for 20 min. 200 μl additional TE was added to the above suspension and DNA was stored at -20 ̊C

In this method of genomic DNA extraction,yeast cells werelysed by mechanical disruption with glass beads. Briefly, yeast cells were harvested after overnight growth in YPD medium, resuspendedin 500 μl waterand transferred toa2 ml microcentrifuge tube.Cells were pelleteddown at 10,000rpm for 1 min. Resulting supernatant was discarded and the pellet was resuspendedin 500 μl Buffer A. The tube was incubated at 65 ̊C for 15 min. After incubation, 500 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) and 0.5 gm of acid-washed glass beads (Sigma # G8772) were addedto the tube. Cells were lysed by three cycles of high speed vortexing withintermittent ice breaksfor 45 secand pelleteddown at 12,000 rpm for 3 minat 4 ̊C.Uppermost aqueous phase was transferred to a 2 ml microcentrifuge tube,500 μl of

Protocol II (Glass bead lysis method)

This quick extraction method was used to isolate genomic DNA which was used as templateto amplify gene of interestor toverify the knock-out. C. glabratacells were grownovernight to saturation in 10 mlYPD medium at 30 ̊C.Cells were harvested at 4,000 rpm for 5 min, resuspendedin 400 μl Buffer Acontaining 50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1%Triton X-100 and 1%SDSand weretransferred to a2 ml microcentrifuge tube. Equal volume ofphenol-chloroform solution was added to the abovesuspensionfollowed byvortexingfor 2-3 minand incubationat 42 ̊C for 30 minwithcontinuous agitation at 800 rpm on thermomixer (Eppendorf). Cell debris was removed bycentrifugation at 12,000 rpm for 5 minand aqueous fraction(~ 350 μl)was transferred to a new 2 ml microcentrifuge tube.0.3 μl RNaseCocktail™(Ambion® # AM2286) containing RNase A (500 U⁄ml) and RNase T1 (20,000 U⁄ml) was added and tubes were incubated at 37 ̊C for 30 min. DNA was precipitated with 2.5 volumesof chilled ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNA pellet was washed with chilled 70%ethanol and semi-dried under air.Pellet was suspendedin 100μlTE (10 mM Tris-HCland 1 mM EDTA; pH 8.0)and stored at -20 ̊C.DNA concentration was determined by recordingabsorbance at 280 nmin Nanodrop (Nanodrop ND-1000, Thermo Scientific).

Protocol

Based on the subsequent use, DNA from C. glabratacells was extracted using three different methodologie

Yeast genomic DNA isolation

C. glabratayeast cells were grown overnight in 5 ml YPD medium at 30 ̊C. An aliquot from the overnight culture was inoculated in 10 ml fresh YPD medium to an initial OD of 0.1. Cells were incubated at 30 ̊C till the cultureOD600was between 0.4 and 0.6. Cells were harvested in a sterile 50 ml centrifuge tube and washed twice with sterile Milli-Q(MQ)water. Washed cells were suspended in 100 μl of 100 mM LiOAc, mixed thoroughly and transferred to a sterile 1.5 ml microcentrifuge tube. A transformation mix containing 240 μlpolyethylene glycol(PEG) (50% (w/v)), 36 μl LiOAc(1 M), 25μl ultrapure single-stranded salmon sperm DNA (2 mg/ml) (Clonetech) was added to 50 μl cell suspension. 50 μltransforming DNA (1μg circular plasmid DNA) was added to the above suspension. Whole mixture was vortexed gently and incubated at 30 ̊C for 45 min. 43 μl DMSO was added to the tubeand incubated at 42 ̊C for 15 min. Cells were collected after centrifugation at 5,000 rpm for 1 min and suspended in minimal medium containing 0.6% Bacto-Casamino acid. Transformation mixture was plated on CAA plates and transformants were selected for uracil prototrophy

Yeast transformation usinglithium acetate (LiOAc) strategy

5-10 ml saturated bacterial culture harboring the desired plasmid was harvested at 5,000 g for 3 min. Plasmid DNAwas isolated using QIAprep Spin Miniprep Kit (Qiagen, USA) or GenElute™ HP Plasmid Miniprep kit (Sigma-Aldrich, USA) as per manufacturer’s instructions

Bacterial plasmid isolation

E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template

Bacterial transformation

suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cellswere immediately snap-frozen in liquid nitrogen and stored at -80 ̊C

A single colony of E.coli DH5-α strain was inoculated in 10ml LB medium and incubated at37 ̊C for overnight. 4 ml of thisovernight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till theOD600reaches to 0.5. Cells were harvested by centrifugation at 2,500 g for 10 min at 4 ̊C and washed gently in 80 ml ice-cold Inoue transformation buffer. Cells were collectedby centrifugation at 2,500 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. Cell

E. coli DH5α ultra-competent cells preparation

All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis

Cultivation of logarithmic-phase cell culture

C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days

Serial dilution spot assay

Yeast cell viability was measured by plating appropriate dilutions of cell cultureonYPD plates at various time intervalsduringgrowth.Cell suspension was diluted in1X PBS. YPD plates were incubated at 30 ̊C for 2-3 daysand total colony forming units(CFUs)were calculated by counting the number of coloniesthat appeared onYPDplatesand dividing that number by anappropriate dilution factor

Yeast cell viability assessment viacolony forming unit (CFU) assay

preparedin appropriate solvents, sterilizedby autoclaving or filtrationand stored at appropriate temperature

For growth analysisof C. glabratastrains, a single colony from YPD or YNBagar mediumwas inoculated in appropriate liquid medium and incubated at 30 ̊C with shaking at 200 rpmfor 14-16 h. This overnight grown culture was used toinoculatetest medium to an initial OD600of 0.1to 0.3.Optical density/Absorbance of the cell suspensionwas measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at600nmat regular time-intervals up to a period of 96 h.Absorbance values were plotted with respect to time. Generation time of yeast strains wascalculated fromthe logarithmic (log) phase of cellgrowth. Growth profilesbetween 4 (t1)and 8 h(t2)time interval wereconsideredfor calculationof generation time usingfollowing formula. Generationtime(G)= (t2-t1) x {log (2)/ [log (Bf/Bi)]}G= Generation time in ht1=Initial timepoint taken for analysist2 = Final timepoint taken for analysisBf= Number of cells at time t2(calculated on the basis of OD600values, wherein1 OD600of C. glabratacorresponds to 2 X 107cells.)Bi= Number of cells at time t1(calculatedas mentioned above)Severalyeast strains used in this study were analysed for their susceptibility to variouschemical compounds,drugsand metal ions. For this purpose, stock solutions were

Growth assayand measurementof generation time

mM final concentration) and pH was adjustedto the desired valueby addition of HCl or NaOH. Medium was sterilized by autoclaving.YNBagar plates ofdifferent pHwereprepared by mixing equal volume of separately autoclaved 4% bacto-agar solution and2X varied pH-adjusted-YNB liquidmedium.All routine sterilization of mediumand solutionswas either carried outby autoclaving at 121 ̊C for 15-20 minat highpressure condition(15 psi)or filtration with 0.2 μmpolyvinylidene fluoride(PVDF) membranefilter unit (Millex®-GV, Millipore).Both yeast and bacterial strains were stored as frozen 15% glycerol stock at -80 ̊Cfor extendedlifetime

C.glabratastrains were maintainedeither on rich YPDor synthetically-defined YNB medium. C.glabratacells were routinely culturedat 30 ̊Cwith shaking at 200 revolutions per min(rpm)unless otherwise mentioned. Forgrowthexperiments, C. glabratastrains were freshly revived on YPDmediumfrom glycerol stocks.Escherichia coliDH5α bacterial strainwasused for plasmid transformation and propagationpurposes and maintained on LB medium.E.coliBW23473 bacterialstrainwas used to rescue Tn7transposon cassette from C. glabrataTn7insertion mutantsand maintained on LB medium. Bacterial strainsharboring plasmids were maintained on LBagar plates supplemented withappropriate antibiotics.For plasmid isolationpurpose,bacterial strains were grown overnight in liquid LB brothcontainingappropriate antibiotics at 37 ̊C with shaking at 200 rpm. Forpreparation of the solid medium, 2%bacto-agar was added to the mediumand autoclaved. To prepare medium of different pH, YNB mediumwas either buffered with citrateor HEPESbuffer (100

Strainsand culture conditions

Microbiological techniques

A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use

Preparation of cell lysate

Experiments involving mice were conducted at VIMTA Labs Limited, Hyderabad in strict accordance withguidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/OS/05). Procedures used in this protocol were designed to minimizeanimalsuffering

Ethics statement

E. colistrains carrying plasmids were inoculated and grown overnight at 37ºC and 200 rpm in LB-liquid medium supplemented with either 50 μg/ml ampicillinor 30 μg/ml kanamycin. Cells were harvested by centrifugation at 2,500g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometerat 280 nmandstored at -20ºC

Plasmid DNA purification

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

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

Strains and culture conditions

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

Harvesting of macrophage-internalized C. glabratacellsfor RNA and protein extraction

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

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

Lysotracker staining

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

Fixing of PMA-treated THP-1 macrophages

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

Cytokines measurement

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

multiplying CFUs with dilution factor and fold-replication was determined by dividing the CFUs obtained at 24 h time-point by 2 h CFUs

Single infection assay

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

Treatmentof THP-1 monocytic cells with phorbol myrsityl acetate

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

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

Cryopreservationand revival of cell lines

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)

Isolation of primary (peritoneal) macrophages from BALB/c mice

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

Cell lines andculture conditions

Animal cell culture methods

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

Isolation of plasmid DNA

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

Total RNA isolation from cultured cells

nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm

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

Transient transfection in adherent cells

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

A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic

Binding reaction

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

The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo

end labelling of the oligonucleotides

Electrophoretic mobility shift assay

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

Immunofluorescence Microscopy

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)

Immunoblotting

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

Protein estimation

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

Extraction of total cellular protein

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

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

Cell cycle analysis

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

Cell growth Assay

Table2.2: Cell types used in the present study

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

Maintenance of cell lines

Cell biology methods

Maintenance of cell lines

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

S. cerevisiae strains were routinely grown either in rich YPD mediumorsyntheticcomplete medium (SC)(Section 2.1.5.1) at 30°C with continuous shaking at 200 rpm unless otherwise stated. In general, S. cerevisiae frozen glycerol stocks were revived on 2% YPD medium by streaking and allowed to grow for 1-2 days. S. cerevisiae strains harbouringaplasmid containingthe URA3geneas the auxotrophy selectionmarker were revived on synthetic complete medium lacking uracil (SC-Ura).To prepare liquid cell culture, a single colony of each S. cerevisiae strain was inoculated either in YPD or SC-Ura medium and grown for 14-16 h. S. cerevisiae strains streaked on plates were sealed with paraffin film (parafilm M) and stored at 4°C for a maximum period of 2 weeks.Protein over expression in yeast was carried in presence of galactose instead of glucose as the carbon source, as the plasmid pYesGex 6p2 carries the GALpromoter under which yeast proteins were expressed

Strains and culture conditions

Cells were plated in a manner that they were 30-50% confluent on the day of transfection.Cells were washed with serum-free medium,and the serum-free medium was added to the cells as per plate size. SiRNA was diluted in the serum-free medium, and oligofectamine was diluted in serum-free media, separately (Table 10). Both the complexes were incubated at room temperature for 5 min. Diluted siRNA wasmixed gently with diluted oligofectamine and incubated at room temperature for 15 min. The final transfection mixture was added dropwise to the cells and mixed properly by gentle rocking. Cells were incubated for 4 hrs.,and the growth medium containing 10% FBS was added to the plates without removing the previous medium. Cells were incubated overnight at 37°C in a CO2 incubator. After overnight incubation, the siRNA transfection was repeated using the same protocol. Cells were harvested after 24-48 hours of second round siRNA transfection. The knockdown was detected bychecking the protein levels throughwestern blotting. (Note: SiRNA transfection is carried out in antibiotic free medium)Table 10: SiRNA transfection methodology

SiRNA

Table 8: Lipofectamine plasmid-transfection methodology

For transfection with Lipofectamine, cells were plated in antibiotic-free medium 24 h before transfection and were transfected at a confluency of 70-80% as per the manufacturer’s protocol. The plasmid of interest was incubated in serum free media,and Lipofectaminewas incubated in serum free media forseparately5minutes. The plasmid and the Lipofectamine mixtures(Table 8)were mixedgentlyand incubated at room temperature for 20 min.;thetransfection mixture was added dropwise to the cells. Transfection media was replaced with the fresh complete medium after 6 hrs.of transfection and cell are harvested after 24 hours

Plasmid transfection using Lipofectamine 2000

Amplified PCR products were run on Agarose gel to check for the amplification ofgene of interests

PCR amplification of the gene of interests was carried out by following the method mentioned in table 4.Table 4: PCR methodology

Polymerase chain reaction (PCR)