Reagents and antibodies
PEGFP-C1-PHF6 plasmid was gift from Ottawa Hospital Research Institute(professor Picketts DJ). NGR1 was obtain from Chen du Pure Chem-Standard Co.,Ltd(Chen du, China). Its chemical structure is shown at Fig. 1A, and the purity is no less than 98%. NGR1 was dissolved in dimethyl sulphoxide (DMSO) which as 100 mM stock solution stored at -20°C, the stock solution was diluted using cell culture medium for the indicated concentration. CCK-8, DAPI and BCA Protein Assay were purchased from Dalian Meilun Biotechnology Co., Ltd(Dalian, China). Sodium dodecyl sulfate (SDS), phenylmethane-sulfonyl fluoride (PMSF), BCA Protein Assay kit, AnnexinV-FITC/ PI reagents were purchased from Beyotime Biotechnology Co., Ltd(Shanghai, China). Primary antibodies against γH2AX, Casepase3, PHF6, GAPDH were purchased from Abcam Company(MA,USA). DMSO, Triton™ X-100 were purchased from Sigma-Aldrich Company(SL,USA). Horseradish peroxidase and fluorescein labeled secondary were obtained from Santa Cruz Biotechnology Co., Ltd (CA, USA). siRNA-PHF6 and Lipofectamine™2000 were purchased from Thermo Fisher Scientific Company(MA,USA)
HeLa cells as cervical cancer cells were obtained from American Type Culture Collection (ATCC, ML, USA). HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, UT, USA) with 10% fetal bovine serum (FBS) (Gibco, YK, USA) and 1% double antibiotics(containing 100U/mL of penicillin and 100μg/mL of streptomycin), and incubated in a humidified atmosphere with 5% CO2 at 37°C.
Cell viablility assays by CCK8 assay
HeLa cells were digested by trypsin and made the cells suspension concentration as 1×105/ml. Inoculated 100μl the cells suspension into a 96-well plate to make per well with 1×104 cells. Cultured them incubator at 37℃ for 4h. After cells adherence, used different concentrations of NGR1 treatment them with 1, 2, 4, 8,and 16mM for 24h and 48h, at the same time each concentration were set 6 replicates. Added 100μl of DMEM medium with 10% CCK8 to each well as a liquid to change. Be carefully, do not has any bubble in the wells to avoid affecting OD value. After incubated its at 37℃ for 4h, the OD value was measured using a spectrophotometer at 450nm. Finally, the cells viability rate were calculated according to the formula:
Cell viability rate = [(As - Ab)/(Ac - Ab)] ×100%
As: absorbance of experimental wells (culture medium containing cells, CCK-8 and NGR1); Ac: absorbance of control wells (culture medium containing cells and CCK-8, but no NGR1); Ab: absorbance of blank wells (culture medium without cells, CCK-8 and NGR1).
Soft agar colony formation assay
Cell colony formation assay was performed as previously described. Briefly, the base agarose was prepared which contained 1×DMEM medium, 0.6% agarose, 10% FBS and 1×antibiotics mixture. Then, the top agarose was prepared which contained 1×DMEM medium, 0.3% low-melting-point agarose, 10% FBS, 1×antibiotics mixture and made the mixture contained 1×103 cells/ml. The top agarose was inoculated on the base agarose for 1ml to make every well contained 1×103 cells, meanwhile, NGR1 was added at 2, 4, 8mM. After being incubated for 12 days, the cells colonies were stained with 0.1% crystal violet for 20 min, and counted them under a microscopy.
Apoptosis rate analysis by flow cytometry
HeLa cells were seed in 6-well plate, when its reached 90% confluence, treated with NGR1 at 1, 2, 4, and 8mM for 12h. Apoptotic cells were quantified by the Annexin V-FITC double staining assay following the manufacturer's instructions. In brief,. Trypsin digested the cells and collected its in plastic tube, each plastic tube with 1×106 cells, washed twice with PBS. added 195 μL Annexin V-FITC binding buffer to suspend the cells, then added 5μL Annexin V-FITC and 10μL PI staining solution. After being mixed gently, the cell suspensions were incubated at room temperature and away from light for 15 minutes, next, placed them on ice. After being filtrated with 300 mesh nylon membrane, the cells were detected with flow cytometry as soon as possible. All experiments were done independently at least three times per experimental point.
Cell cycle analysis using flow cytometry
HeLa cells were treated exactly same as in apoptosis rate analyses described above, In brief, HeLa cells were seed in 6-well plate, when its reached 90% confluence, treated with NGR1 at 0, 2, 4, and 8mM for 24h. After treatment, should be strictly in accordance with the operating instructions, Briefly, the cells (1×106) were collected using trypsinization to digest , washed twice with cold PBS. Cell pellets were fixed in 70% ethanol, washed the cells in cold PBS, resuspended the cells in 0.535 mL of dye buffer containing 10μl RNase(50×) and 25μl PI(25×), incubated the samples in the dark for 30 min at room temperature, and analyzed with a BD Accuri C6 flow cytometer.
Preparation of siRNA and cell transfection
PHF6-siRNA (siPHF6) and nonspecific siRNA(simock) were synthesized from Shanghai Gene Pharma Technology Co. Ltd (Shanghai, China). SiPHF6 sequence is as follows: si-h-PHF6_101: 5ʹ-GGACAGTTACTAATATCTG-3ʹ; si-h-PHF6_102: 5ʹ-GCACGAAGCTGATGTGTTC-3ʹ; si-h-PHF6_103: 5ʹ-CCACTGTGCATTGCATGAT-3ʹ; siRNA(simock): 5ʹ-UUCUCCGAACGUGUCACGU-3ʹ. We had already demonstrated that the interference effect of si-h-PHF6_101 is the best( the date is not show).
The siRNA or PEGFP-C1-PHF6 plasmid and Lipofectamine™2000 with Opti-MEM culture medium which without FBS were mixed gently, then incubated them on ice for 20min. The complexes was added to each cell well. After 6h, the culture medium was exchanged that instead of complete medium to keep cultivating.
Immunofluorescence staining analyses was performed as previously described. Briefly, HeLa cells were fixed with 4% paraformaldehyde for 20min, and then added 0.1% TritonX-100 incubation for 5min. The cells were blocked with 3% bovine serum albumin(BSA) for 1h, and then incubated with anti-PHF6 over nigh at 4°C. After washing, fluoresein-conjugated secondary antibody were incubated to conjugate the anti-PHF6. DAPI was use to label the cell nuclear. The all samples in the same experiments were observed under Olympus Confocal laser scanning microscope.
Western-blotting analysis was performed aspreviously described. Briefly, cells were lysed in RIPA buffer containing 25mM Tris (pH7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1mM PMSF (Sigma), and 1% protease inhibitors cocktail(Thermo Fisher Scientific). The protein in lysate was measured with BCA Protein Assay kit. Equal amounts of protein extracts were loaded on 10% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred them to a PVDF membranes. The membranes were blocked with 5% skim milk for 1h at room temperature and then incubated them with the desired primary antibodies overnight at 4°C. After washing, its were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Detection was performed by using a chemiluminescent ECL advance western blotting detection kit. The primary antibodies used were anti-PHF6, anti-γH2AX and anti-caspase3 antibody. GAPDH served as control. Western-blotting radioactive signal was quantified using Image J.
All experiments were repeated at least three times and the data were presented as the mean ± standard deviation (SD). Student’s t-test was used to compare the differences in all the measurable variables in this study. P values less than 0.05 considered statistical significance.