Cell culture
Cervical cancer cell lines HeLa, SiHa and CasKi, were procured from National Centre for Cell Science, Pune. The cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10%FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 4 mM L glutamine and incubated at 37 °C in a CO2incubator. Cisplatin (P4394) was procured form Sigma Aldrich and diluted in 0.45% NaCl saline solution.
Antibodies
p- H2AX Ser 139 mAb (2577S), p-p53 Ser15 mAb (. 9284S), p-Chk2 Thr68 mAb (2661) Anti-rabbit IgG HRP linked (7074), Anti- mouse IgG HRP linked secondary antibody (7076), Anti- IgG (H+L) F(ab’)2 Alexa Fluor 488 (4412) and Anti-rabbit IgG (H+L) F(ab’)2 Alexa Fluor 555 (Cat. 4413) were obtained from Cell Signalling Technology. MDC1 mouse mAb (1-50) (ab50003) from Abcam and anti human β-Actin (Cat no. SC47778HRP) was purchased form Santa Cruz Biotechnology.
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 (Thermo fisher scientific). The stable cell lines were rigorously selected and maintained in 2.5 μg/ml puromycin and 400 μg/ml G418 for MDC1 knockdown and MDC1 overexpression, respectively.
Cell viability assay
Cell viability assay was done using Cell Titer-Glo luminescent assay kit (Promega, USA) based on quantitation of the ATP . The cells were seeded at a count of 2000 cells per well in a 96 well plate and cisplatin treatment was given to the cells at different concentrations of 5,10 and 20 μM for 72 hours and luminescence reading (Envision, Perkin Elmer).
Clonogenic Survival assays
Cells were treated with cisplatin (10 μM) for 72hours after which they were trypsinized and seeded in 6 cm dishes in triplicate at a count of 1000 cells per plate. After 14 days of incubation the colonies were fixed with methanol and stained with crystal violet (0.5% crystal violet in 20% Methanol, Sigma) and the colony numbers counted. The colonies with ≥ 50 cell count as observed under a stereo microscope were considered for the analysis. Number of colonies derived from the untreated control cells was set as 100% (reference) for comparison. The surviving fraction was calculated by dividing the average number of visible colonies in treated versus untreated dishes.
Western Blotting
Cells were seeded in 6 cm dishes (0.8 × 106/dish) and grown overnight. After treatment with cisplatin cells were lysed in ice-cold RIPA buffer (Sigma, R0278). The soluble fractions of cell lysate isolated by centrifugation at 13,000 rpm for 10 minutes in a micro centrifuge at 4 °C. and 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 incubated with the primary antibody 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 treated with cisplatin (10μM) for 2 hours fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100/ methanol solution for 10 minutes, blocked with bovine serum albumin/ fetal bovine serum for one hour. 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 i.e., 4-6-diamidino-2-phenylindole dihydrochloride (Cat no. NC9524612; VECTASHIELD Antifade Mounting Medium, Fisher Scientific, USA) was used to stain the nuclei.
Flow cytometry analysis of apoptosis
Cells were seeded in 6 cm dishes, incubated for 24 h with 10 μM Cisplatin when 80-90% confluent and stained 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.