Cell culture
Cervical cancer cell lines HeLa, SiHa and CasKi, were procured from National Centre for Cell Science (NCCS), Pune. The cells were cultured in DMEM medium (Thermo Fisher Scientific, USA) with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 4 mM L glutamine. Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Cisplatin (P4394) was procured form Sigma Aldrich and diluted in 0.45% NaCl saline solution.
Stable cell line generation
GIPZ lentiviral MDC1 shRNA (Dharmacon, Inc.) and pCDNA3 MDC1(a kind gift from Prof. Michel Goldberg, Hebrew University, Jerusalem) (Fig S1 and S2) were used to generate the stable cervical cancer cell lines using Lipofectamine 3000 reagent (Thermo fisher scientific). The transformed cells were rigorously selected and maintained in 2.5 μg/ml puromycin (pGIPZ MDC1 shRNA)and 400 μg/ml G418 (pCDN3 MDC1).
Cell viability assay
The cell viability assay was done using Cell Titer-Glo luminescent assay kit (Promega, USA) wherein cells were exposed to cisplatin at different concentrations of 5,10 and 20 μM for 72 hours and luminescence reading was taken (Envision, Perkin Elmer).
Clonogenic Survival assays
The cells were treated with cisplatin at a concentration of 10 μM for seventy-two hours after which they were seeded in 6 cm dishes in triplicate. After 14 days of incubation at 37 °C in a humidified 5% CO2 atmosphere, the colonies were fixed with methanol and stained with crystal violet (0.5% crystal violet in 20% Methanol, Sigma). The number of colonies derived from the untreated control cells was set as 100% (reference) for comparison between mock, MDC1 depleted cells and MDC1 overexpressing cells. The surviving fraction was calculated by dividing the average number of visible colonies in treated versus untreated dishes.
Antibodies
p-Histone H2AX Ser 139 rabbit mAb (Cat no. 2577S), p-p53 Ser15 rabbit mAb (Cat no. 9284S), p-Chk2 Thr68 rabbit mAb (Cat no. 2661) were purchased from Cell Signalling Technology, and mouse anti human β-Actin (Cat no. SC47778HRP) was purchased form Santa Cruz Biotechnology. Anti-rabbit IgG HRP - linked secondary antibody (Cat no. 7074), Anti- mouse IgG HRP linked secondary antibody (Cat. no. 7076), Anti-rabbit IgG (H+L) F(ab’)2 fragment Alexa Fluor 488 conjugate (Cat. 4412) and Alexa Fluor 555 conjugate (Cat. 4413) were obtained from Cell Signalling Technology. MDC1 mouse mAb (1-50) (Cat no. ab50003) was obtained from Abcam.
Western Blotting
Cells were treated with cisplatin and lysed in ice-cold RIPA buffer (Sigma, R0278) containing protease and phosphatase inhibitor. Cell lysates was analysed by SDS-PAGE and western blotting (transfer onto PVDF membrane, Bio-Rad, USA). The membrane was blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight with gentle shaking. Respective secondary antibodies were added the next day and blots were visualised using the Clarity Western ECL luminescent substrate (Cat no.1705061) according to the manufacturer’s instructions (Bio-Rad Laboratories). Beta-actin was used a loading control.
Immunofluorescence assay
Cells were seeded on poly L-lysine coated coverslips and then treated with cisplatin (10μM) for 2 hours at 37 °C. After fixing with 4% paraformaldehyde permeabilized cells with 0.3% Triton X-100/ methanol solution and blocked with FBS. Primary antibody incubation was performed overnight at 4 °C. Cells were stained with TRITC labelled secondary antibody (Alexa Fluor 555 conjugate) for ~2 hours at room temperature. DAPI (Cat no. NC9524612; VECTASHIELD Antifade Mounting Medium, Fisher Scientific, USA) was used to stain the nuclei.
Flow cytometry analysis of apoptosis
Cells were with cyanine 3-conjugated annexin V (AnnexinV-Enzogold) and propidium iodide (PI) using the GFP certified Apoptosis/Necrosis Detection Kit (ENZ-51002, Enzo Biochem, Inc. New York, USA)) as per the manufacturer’s recommendation. Each sample was then subjected to analyses by flow cytometry (FCM) using S3e cell sorter (BIO-RAD, Hercules, CA, USA).
Statistical analysis
GraphPad Prism software (version 5.01) was used for statistical analysis, and p < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). All the tests were either one-way or two-way analysis of variance (ANOVA) followed by multiple comparison post-test.