development. Absorbance was measured at 490 nm, and concentration of glucose production was calculated against glucose standard. Cellulase activity is expressed as micromoles of reducing sugar (glucose) released per minute per 109cells. For plate assay, cell-free culture supernatant of X. oryzaepv. oryzaestrains were inoculated in wells of 0.2% CMC agarose plates. In addition, cellulase assay was also performed by spotting the colony on 0.2% CMC PSA plates. Plates were incubated for 8 to 24 h and stained with congo red to observe the halo formation as described previously (Wood and Bhat, 1988). Extracellular xylanase activity in different X. oryzaepv. oryzae strains was measured using 0.2% 4-O-methyl-D-glucurono-D-xylanremazol Brilliant Blue R (RBB-Xylan) (Sigma-Aldrich) as substrate (Biely et al., 1988)on 1% agarose plates. Xylanase activity is indicated by production of halo around the bacterial colony (Ray et al., 2000). Similarly, for lipase activity p-nitrophenyl butyrate was used as substrate. Lipase activity was calculated by measuring the level of p-nitrophenol released upon hydrolysis of p-nitrophenyl butyrate at 410 nm (Acharya and Rao, 2002). Lipase activity was expressed as micromoles of p-nitrophenol released permin per109cells. For plate assay, colonies were spotted on 1% PSA plates containing 0.5% Tributyrin in 100 mM Tris (pH 8) and 25 mM CaCl2 and halo formation was observed for lipase activity

For extracellular enzyme assays, X. oryzaepv. oryzae strains were grown in PS, MM9 and XOM2 media to an OD of 0.6, and centrifuged at 12,000 g for 10 min to collect the supernatant. The supernatant was taken as an extracellular fraction and cell pellet was plated by dilutionplating to get the CFUs per milliliter of culture. Extracellular cellulase activity was measured using phenol-sulphuric acid (H2SO4) method, which measures pentoses and hexoses (concentration of glucose released) upon cellulase activity (DuBois et al., 1956). Briefly, a specific amount of supernatant was taken and volume was adjusted to 300 μl by adding 50 mM acetate buffer (pH-5.4). To this, 1% carboxy methyl cellulose (CMC) substrate solution was added and mixed well. This mixture was incubated at 28°C for 30 min, and the reaction was stopped by adding 1 ml ice-cold ethanol. Solution was mixed well, kept on ice for 5 min and centrifuged at 12,000 g for 5 min. Supernatant was recovered and 5% phenol was added to it, mixed well followed by adding 1 ml H2SO4. The tube was incubated at RT for 20 min for co

Extracellular enzyme assays

200 rpm in LBbroth supplemented with appropriate antibiotics (plasmid antibiotic marker). Cells were harvested by centrifugation at 12,000 g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep ormidiprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometer at 280 nm and stored at -20°C

E.colistrains carrying plasmids were inoculated and grown overnight at 37°C and

Plasmid DNA purification

A microtipful cells of bacterial strain from appropriate medium was resuspended in 20 μl sterile water and incubated at 98°C for 10 min for cell lysis. 2 μl of heat-lysed cell suspension was used as template in 25 μl PCR reaction

Xanthomonasand E.colicolony PCR

and finally resuspended in 100 μl sterile water. Bacterial cell suspension was aliquoted in 20 μl volume. The above procedure was followed for all the three strains and cell suspension of three different strains were mixed together in 1:1:1 ratio. For conjugation to occur, 20 μl of the above mixture was spottedon the LB agar plate and incubated at 37°C for 12-16 h. Next, the conjugation drops were streaked on LB agar plate containing appropriate antibiotics to select the S17-1 recipient containing recombinant plasmid.S17-1 was directly conjugated with Xanthomonasstrain. S17-1 strain containing recombinant plasmid (3 ml) and recipient Xanthomonasstrain (100 ml) was grown overnight with appropriate antibiotics. Cells were harvested and washed thrice as mentioned earlier. Xanthomonasstrain was finally dissolvedin 600-700 μl sterile water and S17-1 strain was dissolved in 3 ml sterile water. 50 μl Xanthomonascell suspension and 10 μl S17-1 cell suspension were mixed together and 20 μl was spotted on PS agar plate. After 40 h of incubation at 28°C, each conjugation drop was dissolved in 400 μl water separately and plated on PS agar medium with rifampicin (counter-selectable marker) and plasmid specific antibiotics for specific selection of Xanthomonascolony with recombinant plasmid

Since compatible conjugation does not exist between Xanthomonasand E.coliDH5α strain.Therefore, upon getting the appropriate clones in DH5α, conjugation was performed with S17-1 (recipient strain) and PRK600 (helper strain). All the three strains (DH5α with clone, S17-1 and PRK600 strain of E.coli) were grown overnight at 37°C with constant shaking at 200 rpm in 3 ml LB broth. Cells from 1 ml overnight grown cultures were harvested by centrifugation followed by three washes with s

Xanthomonas conjugation

shaking at 200 rpm. 1% of overnight grown culture was inoculated in 100 ml fresh PS medium and grown to obtain log-phase culture. Log phase Xanthomonas culture was kept on ice for 10-15 min, aliquoted in 50 ml pre-chilled centrifuge tubes and centrifuged at 4000-5000 g at 4°C for 10 min. Supernatant was discarded and pellet from each tube was gently resuspended in 10-20 ml sterile chilled water. Next, cells were harvested by centrifugation at 4000 g at 4°C for 10 min and supernatant was discarded. Harvested cells were washed twice and finally resuspended in adequate amount of prechilled sterile water. 100 μl of cell suspension was aliquoted in sterile 1.5 ml microcentrifuge tubes and kept on ice. For transformation, Xanthomonaselectrocompetent cells and appropriate amount of plasmid DNA was mixed, and kept on ice in laminar hood. This mixture was added to 1 mm electroporation cuvettes (Biorad) and tapped gently to allow the cells to settle properly in order to avoid air bubbles. Competent cells were electroporated (1800 V, 25 μF, 200 Ω, 1mm cuvette) followed by immediate addition of fresh PS broth in the cuvette, mixed properly and taken in the microcentrifuge tubes. Microcentrifuge tubes containing transformed cells were incubated at 28°C for 2 hours with continuous shaking for recovery. After recovery, cells were plated on specific medium with appropriate antibiotics and incubated in 28°C plate incubator

For electrocompetent cell preparation, single colony of desired Xanthomonasstrain was inoculated in 5 ml PS medium and grown overnight at 28°C

Xanthomonastransformation

E.coliDH5α strain was transformed with plasmids carrying appropriate inserts to generate clones, and Xanthomonas deletion strains. Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 5 μl ligated plasmid was added to 100 μl ultracompetent cells and incubated on ice for 30 min. Next, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred on ice for 2-3 min. Next, 1 ml LB medium was added and cells were allowed to recover for 1 h on a shaker incubator set at 37°C. After the recovery, cells were centrifuged at 3000 g for 3 min. Medium supernatant was discarded and cells were resuspended in 100 μl fresh sterile medium. Cells were plated on LB agar containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 h

E.colitransformation

A single colony of E.coliDH5α strain was inoculated in 5 ml LB medium and incubated at 37°C for overnight. 1% of overnight grown culture was inoculated in 500 mlfresh LB medium and incubated at 37°C for 2-3 h till the OD600 reached to 0.4-0.5. Culture was chilled on ice for 5 min followed by centrifugation at 3000 g for 15 min at 4°C. Harvested cells were washed gently with 200 ml ice-cold TFb-I buffer. Cells were collected by centrifugation at 3000 g for 5 min at 4°C and gently resuspended in 20 ml ice-cold TFb-II buffer. Bacterial cell suspension was kept on ice for 15 min and was aliquoted in 100 μl volumes in chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E.coliultracompetent cells

Xanthomonas strains were grown in PS medium for 14-16 h at 28°C with continuous shaking at 200 rpm. 1 ml of bacterial cultures were ten-fold serially diluted in water and 100 μl volume of each dilution was plated on PS agar plates to get the colony forming units (CFUs). Similarly, 5 μl volume of each dilution was spotted on PS agar plates containing different concentration of streptonigrin and different detergents for intracellular iron and membrane sensitivity assay, respectively. Plates were incubated at 28°C and images were captured after 2-8 days of incubation depending upon m

Serial dilution plating and spotting assay

For growth analysis of Xanthomonasstrains, a loopful of bacterial colony was inoculated in appropriate broth medium and grown for 14-16 h. 0.2% of overnight grown culture was used to inoculate the test medium (for iron limitation, PS with 50 or 100 μM of 2,2’-dipyridyl, and for iron supplementation, different concentrations of either FeCl3or FeSO4was added). Cultures were transferred to a shaker incubator set at 28°C and 200 rpm. Absorbance of cultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences)at 600 nm at regular time-intervals till 48 h. Absorbance values were plotted with respect to time and generation time was determined from the logarithmic (log) phase of bacterial growth using the following formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf = Absorbance at time T2(Final OD)Ni= Absorbance at time T1(Initial OD)

Growth analysis and determination of generation time

Xanthomonasstrains were routinely grown in rich PS medium, at 28°C with continuous shaking at 200 rpm unless otherwise stated (New Brunswick Scientific, Innova 43, U.S.A.). In general, Xanthomonas frozen glycerol stocks were revived on PSA medium by streaking,and allowed to grow for 3-4 days. To prepare liquid culture, a loopful of each Xanthomonasstrain was inoculated in PS medium and grown for 24-30 h. Xanthomonasstrains on plates were stored at 4°C for a maximum period of 1 week. For growth of X. oryzaepv. oryzaein Minimal (MM9); (Kelemu and Leach, 1990)and XOM2 media (minimal media which induces hrp genes in X. oryzaepv. oryzae),(Tsuge et al., 2002), first the strains were grown in PS medium to a cell density of 109cells/ml and then centrifuged at 5000 g to concentrate the cells and washed twice with sterile water to remove media components sticking to the cells. Washed cells were inoculated in MM9 and XOM2 medium and grown for overnight.Escherichia coliDH5α,used for cloning purposes, was revived on LB medium containing nalidixic acid and grown at 37°C with continuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to grow the bacterial strains carrying plasmids. For plasmid purification, bacterial strains were grown overnight in LB broth medium containing suitable antibiotics.Antibiotics were used at a final concentration of 50 μg/ml rifampicin, kanamycin, streptomycin and trimethoprim; 100 μg/ml ampicillin; 25 μg/ml nalidixic acid; 10 μg/ml cephalexin, chloramphenicol and gentamicin; 20 μg/ml cyclohexamide and 5 μg/ml tetracyclin

Strains and culture conditions

Microbiological methods

Experiments involving mice were performed at the CDFD animal facility, VIMTA Labs Ltd., Hyderabad, India(www.vimta.com) in strict accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/CDFD/05). Procedures used in this protocol were designed to minimize animal suffering

Ethics statement

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Yeast genomic DNA isolation

C. glabratastrains were routinely grown in rich YPD medium or synthetically defined YNB medium, or YNB medium supplemented with CAA, unlessstatedotherwise.To obtain overnight grown liquid cultures, C. glabratacells were inoculated in appropriate medium and incubated at 30°C under constant agitation (200 rpm) to maintain proper aeration.To revive the frozenstocks,about one tipfull of frozen culture was streaked either on YPD-agar or on CAA-agar medium. In general, frozen stocks of C. glabratastrains were revived on YPD-agar medium.However,C. glabratastrains harbouring plasmidscontainingURA3as a selectable marker were revived on CAA-agar medium. After streaking, plates were allowed to grow for 24-48 h at 30°C and were stored at 4°C for a maximum period of two weeks. For long term storage, freezer stocks of C. glabratastrainswere prepared in 15% glycerol and stored at -80° C.Escherichia colistrain DH5αwas revived on LB-agar medium from frozenstock and incubated at 37°C for 14-16 h. DH5α strainwas used for transformation purpose and maintaining plasmids. Bacterial strains harbouring plasmids containing selection markerswere revived on LB-agar medium supplemented with appropriate antibiotics.Bacterial liquid cultures were either grown in LB broth or LB broth containing suitable antibioticsand incubatedin a shakerincubator set at 37°C, 200 rpm for 14-16 h. For preparation of bacterial frozenstocks, 1 ml overnight grown bacterial culture was added to500 μl of 50% glycerolto obtain final concentration of ~16 % glyceroland stored at -80°Cuntil use

Strains and culture conditions

PMA-treated THP-1 cells were seeded toa24-well tissue culture plate to a cell density of 1 million cells per well and allowed to grow for 12 h. After12 hincubation,spent medium was replaced with fresh pre-warmed RPMI-1640 medium andcells were allowed to recover for 12 h before use.C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 ml of theseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x107cells/ml. 50 μl of this cell suspension was used for infection to a MOI of 1:1. Two hours post infection, wells were washed thricewith 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed RPMI-1640 medium was added. Plates were incubated under tissue culture conditions at 37°C and 5% CO2for 24 h. Supernatants were collected in 1.5 ml microfuge tubes, centrifuged at 3,000 rpmto remove the particulate matter, if any, and stored at -20°C until use. Estimation of different cytokines were performed using BD OptEIA ELISA kits as per the supplier’s instructions

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

To perform survival analysis of C. glabratacells in macrophages, PMA-treated THP-1 cells were seeded to 24-well tissue culture platesto afinal celldensity of 1 millionper well. C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 mloftheseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x106cells/ml. 50 μl of this cell suspension was used to infect PMA-activated macrophages to a MOI (multiplicity of infection) of 0.1. Two hours post infection, THP-1 cells were washed thrice with 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed complete RPMI-1640 medium was added.At different time points, infected THP-1 macrophages were osmolysed with1 ml sterile water. Post lysis,lysates were collected by scraping the wells using 1 ml microtip. Lysates were diluted in 1X sterile PBSand appropriate dilutions were plated onYPD-agar plates. Plates were incubated at 30°C for 24-48 h and colony forming units (CFUs) were counted. Final CFUs/ml were determined by multiplying CFUs withappropriate dilution factor and percentage phagocytosis was calculated by dividing CFUs obtained at 2 h post infection by total numberofyeast cells used for infection. Fold replication was calculated by dividing the CFUs obtained at 24 h post infection by CFUs obtained at 2 h post infection

THP-1 macrophageinfection assay to monitor the intracellular survival and replication of C. glabrata

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

PMA (Phorbol myristateacetate) treatment of THP-1 monocytic cells

For cryopreservation of THP-1 and Lec-2 cells, 5-6 million cells wereresuspendedin 0.5 ml of eithercommercially procuredcell preservation medium from GIBCO(12648010)or complete medium supplemented with 10 % fetal bovine serum and 10 % DMSO.Cells were initially kept inanisopropanol bath and werelatertransferred to -70°C freezer. After 2-3 days, frozencells were transferred to liquid nitrogen container till further use. To revive the cells, frozenstockswere taken out of the liquid nitrogen container and immediately transferred to water bath set at 37°Cfor thawing. When freezing medium has thawed completely, cells were transferred to a 100 mm cell culture dishcontaining 12 ml completemedium and incubated under tissue culture conditions at 37°C and 5% CO2for 12 h. Afterincubation, medium was replaced by 12 ml fresh pre-warmed medium and incubated under tissue culture conditions till they reached 70-80% of confluencebefore splitting

Cryopreservation and revival of cell lines

Human monocytic cell line THP-1 (ATCC TIB-202TM), derived form 1 year old acute monocytic leukemia patient,wasused to perform single cell infection assays. Differentiation of these THP-1 monocytic cellsto phagocytic cells was induced by using phorbol myristateacetate (PMA)(16 nM). Lec2 (ATCC CRL-1736TM),an ovary epithelial cell line,which has beenderived fromtheChinese hamster Cricetulus griseus, wasused to determine the adherence of C. glabratacells. THP-1 and Lec-2 cells were routinely cultured and maintained in RPMI-1640 and α-MEM media, respectively, supplemented with10 % heat inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml) andstreptomycin (100 μg/ml). Cultures were maintained in cell culture incubator (Thermo scientific)at 37°C and 5% CO2. Cultures were split after every 2-3 daysandspent medium was replaced with fresh pre-warmed medium. For splitting, cells were harvested at 1,000 rpm for 2-3 min, spent medium wasdiscarded and cell pellet was resuspended in 4-6 ml of pre-warmed medium. Cell density was determined by using hemocytometer. A total of3-4 million cells were seeded in a 100 mm cell culture dishcontaining 12 mlfresh medium

Cell lines and culture conditions

Animal cell culture methods

HEK293T packaging cellswere seeded at 30-40% confluency in 60 mm dishes. After 24 h, cells were co-transfected with three plasmids required for viral production i.e. VSV-G, psPAX2 (Addgene plasmid # 12260) and pLKO.1-puro-non-targeting and shIP6K1 clones using polyethyleniminereagent(PEI) and incubated at 37°C and 5% CO2 for virion formation. After 48 h, viral particles were harvested by collecting supernatant and filtered througha 0.45 μm syringe filter unit. Viral stock was aliquoted and stored at -80°C for further use. Viral titer was approximated on the number of cells plated for the production of lentivirus. Calculations were done as per Cell Bio Labs instruction. 2 x 106cells will yield 107infectious units/mL. All necessary precautions were taken while generating lentiviral particles such as wearing mask, double gloves, and sterile filter tips. All the consumables used were bleached (1% sodium hypochlorite solution) at least 1 h before being discarded

Generation of lentiviral particle containing shRNAagainst human IP6K1-

cells convert MTT to insoluble purple formazan dye crystals which can besolubilized using detergent or DMSO and measured by colorimetry. At each time point mediacontaining MTT dye (250 μg/mL) was added to each well and incubated at 37ºC. After 2 h cells were lysed with 200 μL DMSO for 15 min on a rocker at room temperature to solubilize the formazan and absorbance was monitored at 570 nm using the EnSpire multimode plate reader (PerkinElmer). Doubling time was determined by plotting log [A570] vstime and conducting a linear regression analysis using GraphPad Prism 5

Cells were seeded in 24 well plates at 10% confluence in triplicates and allowed to grow for different lengths of time. Spent medium was replaced with fresh medium every 48 h. Cells were incubated for different lengths of time ranging from 24 h to 120 h, to allow them to grow and cell survival was monitored by the MTT assay. Metabolically active

Cell proliferation assay