Cell lines and reagents
Ishikawa (Sigma-Aldrich; Merck KGaA) and AN3CA (American Tissue Culture Collection) cells were cultured at 37˚C in a humidified incubator with 5% CO2 in Eagle's Minimum Essential Medium with 10% FBS for <3 weeks.
Cisplatin resistant Ishikawa and AN3CA cells were generated by incubating WT cells in medium with 2.5 µM cisplatin for 4 weeks, subsequently followed by 5 and 10 µM cisplatin for 4 weeks at each concentration. Doxorubicin resistant Ishikawa and AN3CA cells were generated by incubating WT cells in medium with 0.5 µM doxorubicin for 4 weeks, subsequently followed by 1 and 2 µM doxorubicin for 4 weeks at each concentration.
Cisplatin and doxorubicin were purchased from Sigma-Aldrich.
Analysis of cell viability
EC were seeded in 96-well plates at a density of 5x103 cells/well in triplicate. After 24 h, cells were treated with cisplatin or doxorubicin for 48 h and the cell viability was measured using MTT assay. Briefly, 10 μl MTT (Sigma-Aldrich; Merck KGaA) solution was added to each well, and the plates were incubated for 2.5 h at 37˚C. The absorbance was measured at 490 nm using a Microplate spectrophotometer (BioTek Instruments, Inc.).
NHEJ reporter assay
A total of 10 μg of NHEJ reporter plasmid was linearized using NheI and purified using the QIAquick Gel Extraction kit according to the manufacturer’s instructions (Qiagen Corporation). 1 μg of purified plasmid was transfected into EC cells using Lipofectamine® 2000 according to the manufacturer's protocols (Thermo Fisher Scientific, Inc.). Cells with chromosomally integrated reporter were selected by incubating in medium with 1 mg/ml geneticin for 2 weeks. To measure NHEJ efficiency, stable reporter cells were seeded at 2x105 cells/ml in a 6-well plate and allow attachment for 24 h. To start the NHEJ, 1 µg of I-SceI plasmid was transfected into stable reporter cells. Cells were incubated for 48 to generate DSB and repair. Cells were harvested and green fluorescent protein (GFP) positive cells, which indicate successful DSB repair, were count using BD FACSCelesta™ Flow Cytometer (BD Biosciences).
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted using the RNeasy Mini Kit (Qiagen Corporation) and was reverse transcribed using the TaqMan™ Reverse Transcription Reagents (ThermoFisher Scientific). Subsequently the Fast SYBR™ Green Master Mix (ThermoFisher Scientific) was used for qPCR to detect the mRNA levels of RNF8, according to the manufacturer’s instructions using a Real-Time PCR system (Eppendorf Thermal Cycler Eco; Eppendorf). The following primers were used: RNF8 forward, 5′-ATTAAGTTGCGCGAGAGGAA-3′ and reverse, 5′-AGCTCGTTCTCCAGCAAGTC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′
Studies involving patient samples were approved by the China-Japan Union Hospital of Jilin University, and all patients provided informed consent, in accordance with the Declaration of Helsinki.
CRISPR-Cas9-mediated RNF8 deletion
A pool of 3 plasmids encoding Cas9 coding gene and RNF8 guide RNA (cat. no. sc-401909; Santa Crus Biotechnology, Inc.) or CRISPR/Cas9-Ctr Plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.) was transfected into AN3CA Cis-R and AN3CA Dox-R cells using Lipofectamine® 2000. Cells were harvested and seeded in 96-well plate at densities of 100, 200 and 300 cells/ml and incubated for 2 weeks or until single colonies form in each well. Single clones were picked and expanded. RNF8 expression was assayed using western blot analysis with RNF8 antibody (cat. no. sc-271462; Santa Crus Biotechnology, Inc). 2 RNF8 KO clones were randomly picked for further studies.
Western blot analysis
Cells were pelleted, resuspend and vortexed in RIPA lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP40). Cell lysate was centrifuged for 20 minutes at 12,000 x rpm at 4°C and the supernatant was retained, following which the protein concentration of the lysate was determined. Cell lysate samples were denatured using SDS-PAGE sample buffer and separated using SDS-PAGE. Separated samples were transferred to nitrocellulose membrane (BioRad Laboratories, Inc.). Membrane was blocked with 3% BSA in 1X PBST buffer (1xPBS with 0.1% Tween-20). The membrane was ten incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The protein signals were detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and the proteins were visualized using the SuperSignalTM west pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Inc.). The following antibodies were used: anti-actin (cat. no. 3700; Cell Signaling Technology, Inc.), anti-Ku80 (Cell Signaling Technology, Inc.), anti-RNF8 (cat. no. sc-271462; Santa Crus Biotechnology, Inc).
Laser microirradiation and Immunofluorescence staining
AN3CA Cis-R cells were seeded at 3x105 cells/ml on glass-bottomed culture dishes (MatTek Corporation), following which cells were stained with Hoechst33342 (10µg/ml) for 1 h. Cells were then exposed for 15 s to the 405 nm laser microbeam with a Micropoint Ablation System (Photonics Instruments, St. Charles, IL, USA), which is focused by a 60x oil immersion inverted microscope objective. Cells were then incubated at 37˚C in a humidified incubator with 5% CO2 for 4 h. After washing with PBS, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized in 0.5% triton X-100 solution for 5 min at room temperature. Cells were blocked with 3% BSA in wash buffer (1xPBS with 0.02% Tween-20) for 30 min and incubated with primary antibody for 2 h. Subsequently, samples were washed with wash buffer and incubated with secondary antibody for 45 min. Nuclear DNA was stained with DAPI staining solution. The coverslips were mounted, and signals were visualized by a fluorescence microscope (Nikon ECLIPSE E800).
All the animal experiments were authorized by the Laboratory Animal Care and Ethical Committee of the China-Japan Union Hospital of Jilin University, and were performed following the Guide for the Care and Use of Laboratory Animals (8th edition, 2011, National Academies Press (US)).
BALB/c nude mice (female, 4-week-old, 18 ± 2 g) were purchased from Charles river (Beijing, China). 5x106 AN3CA Cis-R or AN3CA Cis-R-KO cells were subcutaneously injected in the right flank to generate tumor xenograft model. Tumor size was assessed by measuring tumor diameters with Vernier calipers twice a week. Tumor volume is calculated according to the formula, volume=length x width2/2. Treatments were given when the tumors were approximately 100 mm3 in volume. Mice were treated with vehicle or cisplatin (8 mg/kg/3 day) intraperitoneally for 2 weeks. Tumor volume and body weight were measured every 3 days for 3 weeks.
GraphPad software (Prism; v7.0) was used to create and analyze all the graphs. A Student’s t-test was used for comparison of two groups. A one-way or two-way ANOVA analysis with a Bonferroni post-test was used to compare multiple groups. P<0.05 was indicated to indicate a statistically significant difference.