Antibodies, Plasmids and Chemicals
Liver cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in DMEM medium (Thermo Fisher Scientific, Shanghai, China) supplemented with 10% fetal bovine serum at 37℃ with 5% CO2 and 95% humidity. The following antibodies were used for Western blotting: WIP1 (sc-376257) to detect human samples from Santa Cruz (Shanghai, China); WIP1 (A6204) to detect mouse samples from ABcolonal (Wuhan, China) ; cleaved PARP1(9541), cleaved-caspase3 (9661) and beta-Actin (4970) from Cell Signaling Technology (Shanghai, China); phospho-Histone H2ax (s139) (ab81299) from Abcam (Shanghai, China); ki67 (ER1802-31) from Huabio (Hangzhou, China). WIP1 plasmid was kindly provided by Prof. Zhenyu Ju at Hangzhou Normal University. GSK2830371, Diethylnitrosamine (DEN) and TCPOBOP were purchased from Sigma-Aldrich (Shanghai, China). Olaparib (HY-10162) and Veliparib (HY-10129) were purchased from MedChemExpress (Shanghai, China). Other reagents and chemicals don’t list here are commonly commercial available.
siRNAs and Plasmids Transfection
siRNAs mentioned in this article were synthesized by Gene Pharma Company (Shanghai, China), and transfected into cells with LipofectamineTM RNAiMAX transfection reagent (Thermo Fisher Scientific) at a final concentration of 20-50nM. All siRNAs sequences used were listed in Supplemental Table 1.
For plasmid transfection, cells were seeded overnight in 6 well plates, 2 μg of plasmids were transfected with X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science, Shanghai, China). The mock vector was used as the negative control. Cells were harvested for indicated analysis after 48-72 hours later.
To consistently knock down WIP1, cells were seeded overnight in 6-well plates and infected with lentivirus contain pLKO.1-scramble (shNC) or pLKO.1-shWIP1 (shWIP1). Stable cells were screened by puromycin. Knockdown of WIP1 was verified by Western blotting, and the constructed stable cells were sent out for cell proliferation in vitro and in vivo respectively. Sequences used were listed in Supplemental Table 2.
Cell growth assay
Cell growth assay was applied with the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit (Promega, Beijing, China). Briefly, the cells were seeded into a 96-well plate overnight and treated as indicated, and the MTS reagents were added to each well. Cell viability was measured following the manufacturer’s instruction. Samples were prepared in triplicates, and the cell viability was determined as the mean ± s.d.
Plate colony-formation assay
Stable knockdown cells were screened by puromycin before colony-formation assays with monolayer cultures. PLC/PRF/5 cells were seeded at 300 cells/well in 6 well plates. Hepa1-6 cells were seeded at 150 cells/well in 6 well plates. After 14 days of culture, cell colonies were counted after staining with 0.5% crystal violet.
HCC cells treated as indicated were harvested with Radio immunoprecitation assay (RIPA) buffer and then were quantitated by BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Lysates were resolved by SDS-PAGE, transferred to PVDF membrane and incubated with the primary antibodies at 4℃ overnight, then washed with TBS-T (TBS with 0.1% of Tween-20) and incubated with HRP-conjugated second antibodies (Jackson ImmunoResearvh Laboratories) at RT for 2 hours, after that, the membranes were test with FDbio- Femto ECL (FD8030, Fudebio, Hangzhou, China), and pictures were processed with Amersham Imager 600 system (GE Healthcare Life Sciences, Shanghai, China).
Immunohistochemistry (IHC) staining
Formalin-fixed and paraffin-embedded human liver tissue sections were first sent for hematoxylin-eosin (HE) staining and subsequently immunostained with anti-ki-67 antibody using microwave antigen retrieval in 0.01M pH6.0 citrate buffer. After washing, signal was detected using suitable HRP labeled second antibody with DAB as the chromagen (Dako, Denmark).
Immunofluorescence and microscopy
Cells were seeded on coverslips overnight and treated as indicated. Briefly, the cells were fixed with cold methanol for 10 min, permeabilized in 0.25% Triton X-100 for 10 min and blotted with 3% BSA (bovine serum albumin; diluted in PBS) for 30 min. The appropriate primary antibodies were diluted with 3% BSA and incubated with the cells at 4 °C overnight. Then, the coverslips were washed three times with 0.5% PBS-T (PBS with 0.5% Tween-20), incubated with the appropriate secondary antibodies for 1 h at room temperature, washed and sealed with mounting medium including DAPI. Images were captured on microscope (Olympus, Japan).
Den induced mice liver cancer model
Wip1 KO mice were kindly provided by Prof. Lawrence A. Donehower26. The mice were maintained and treated under specific pathogen-free conditions. To induce hepatocellular carcinogenesis, 100 mg/kg DEN was i.p. injected into 4-weeks-old male mice, and after 2 weeks, 3 mg/kg TCPOBOP (Sigma) was i.p. injected into the mice every other weeks for 8 times. Ten months after the DEN injection, mice were euthanized. The liver tissues were collected and divided, one half was immediately frozen in liquid nitrogen and stored at -80℃ until sent for quantitative real-time RT-PCR and Western blot analysis, another half was fixed with 4% formaldehyde immediately and sent for HE staining. The numbers of liver tumor (diameter>2mm) of each mice was counted and student’s t test was performed for statistical analysis.
Tumor-formation assay in C57BL/6J mice
Male C57BL/6J mice (6-8 weeks of age) were obtained from Shanghai Laboratory Animal Center and housed in the laboratory-animal research center of Zhejiang University. Cultures of Hepa1-6-shNC or Hepa1-6-ShWIP1 cells were resuspended with PBS. Mice were randomized divided into two groups (n=5/group), and 1×106 of cells were subcutaneous injected into each mouse. After 7 days, growth of implanted tumors was monitored using Vernier calipers every 2 days. Tumor volume (cm3) = 0.5 × Tumor length× Tumor width2. All mice were sacrificed after 17 days.
Tumor-formation assay in nude mice
Male BALB/c nude mice (n=56, 6-8 weeks of age) were obtained from Shanghai Laboratory Animal Center and housed in the laboratory-animal research center of Zhejiang University. PLC/PRF/5 cells were resuspended with PBS, and 3×106 cells were subcutaneous injected in to each mouse. After 7 days, mice were randomized divided into 4 groups (n=7) and oral treated with Blank/GSK2830371 (100mg/kg) /Olaparib (50mg/kg)/ GSK2830371（50mg/kg）+ Olaparib(25mg/kg) three times a week. For another experiment, 3×106 PLC/PRF/5 cells were subcutaneous injected in to each mouse. After 7 days, mice were randomized divided into 4 groups (n=7) and oral treated with Blank/GSK2830371 (100mg/kg) /Veliparib (100mg/kg)/ GSK2830371（50mg/kg）+ Veliparib(50mg/kg) three times a week. For each experiment, the growth of implanted tumors was monitored using Vernier calipers three times a week: Tumor volume (cm3) = 0.5 × Tumor length × Tumor width2. All mice were sacrificed after 23 days.
To identify the association of WIP1 expression with DNA damage response, global gene expression profiles in paired human HCC tissues was obtained from GEO database (GSE57957), and was analyzed with Gene Set Enrichment Analysis(GSEA) using GSEA 3.0 software (http://www.broadinstitute.org/gsea/), the Gene Set of DNA double strand break response and Mismatch Repair from MsigDB was employed for GSEA27. And the survival analysis and correlation analysis were performed via GEPIA2.0 (http://gepia2.cancer-pku.cn/). The TMB data( Tumor mutation burden) of HCC patients was download from TCGA database, and was calculated via maftools R packge28.
An independent Student’s t test was performed to analyze the assay results. Pearson analysis was performed to analyze the correlation. p value < 0.05 was considered statistically significant. Results are expressed as mean ± SD as indicated. All experiments were repeated at least three times.