Fig. 7

HCR depletion causes mitotic defects, DNA damage, and decreased tumor proliferation. A After releasing from double-thymidine arrest for the indicated time, parental HeLa cells and HCR-KO HeLa cells were fixed and stained with PI (DNA staining) for flow cytometry. The DNA content in cells is diploid (2N) in the G1 phase and becomes tetraploid (4N) from S to G2/M phase. When mitosis ends, the DNA content in the cell should revert from 4N to 2N. The cell cycle results were analyzed and plotted based on the DNA content in cells. A total of 10,000 cells were counted per group. B Negative control, astrin, HCR, and CEP72 siRNA-treated HeLa cells were treated with 100 ng/ml nocodazole for 16 h to be arrested in M phase and co-stained with gamma-tubulin (green), alpha-tubulin (red), and DAPI (blue). Cells (n = 100 each group) were counted from three independent experiments. Error bars represent the mean ± SD; *P < 0.05 (Student’s t test). C Negative control, astrin, HCR, and CEP72 siRNA-treated HeLa cells were treated with 100 ng/ml nocodazole for 16 h to be arrested in M phase and were analyzed by immunoblotting with antibodies to securin, separase, HURP, cyclin B1, and beta-actin, and the relative protein levels of securin and cleaved separase were analyzed statistically. Error bars represent the mean ± SD of three independently performed experiments (n = 3); *P < 0.05 and **P < 0.01 (Student’s t test). The individual data values were provided in Additional file 6: Raw Data. D Negative control and HCR siRNA-treated HeLa cells were stained with DAPI (blue). The quantified analysis was based on the percentage of the cells containing micronucleus. Cells (n = 100) were counted from three independent experiments. Each bar represents the mean ± SD; ***P < 0.001 (Student’s t test). Arrow represents the micronuclei. E Negative control and HCR siRNA-treated HeLa cells were co-stained with pATM (green) and DAPI (blue). The quantified analysis was based on the percentage of pATM-positive cells. Cells (n = 100 each group) were counted from three independent experiments. Error bar represents the mean ± SD; **P < 0.01 (Student’s t test). F Negative control and HCR siRNA-treated HeLa cells were co-stained with gamma-H2AX (green) and DAPI (blue). The quantified analysis was based on the percentage of gamma-H2AX-positive cells. Cells (n = 100 each group) were counted from three independent experiments. Error bar represents the mean ± SD; **P < 0.01 (Student’s t test). G Negative control and HCR siRNA-treated HeLa cells were analyzed by immunoblotting with antibodies to pCHK2, CHK2, and beta-actin. H Colony formation assays of parental HeLa cells, HCR-KO cells, and astrin-KO cells. I Parental HeLa cells, HCR-KO cells, and astrin-KO cells (1 × 10⁶) were transplanted in the athymic mice, and tumor sizes were measured every 3 days after the formation of a measurable tumor. Error bars represent the mean ± SD for different animal measurements (n = 5 each group); P < 0.01, one-way ANOVA for tumor weight analysis and two-way ANOVA for tumor size analysis. The individual data values were provided in Additional file 6: Raw Data