Figure 6: Biological consequence of the PbR mutation in response to replication fork stress.
(a) The experimental design to test the function of PbR. GFP-tagged Chk1 vectors were transfected into monkey COS7 cells. Endogenous monkey Chk1 is knocked down (KD) by monkey-specific siRNA. (b) IB analysis of Chk1 activation. COS7 cells stably transfected with empty vector-GFP (EV-GFP), wild type (wtChk1-GFP) or PbR-mutant Chk1 (PbRC/AChk1-GFP) vectors were treated with siChk1 or non-target siRNA (nt-siRNA). At 3 days after siRNA treatment, cells untreated (−) or treated with 2 mM HU for 3 h were analysed for the indicated proteins at 0 and 3 h after HU removal. COS7 cells (wtChk1-GFP+nt-siRNA) were used as controls. PARP1 and β-tubulin were used as loading controls. (c) RDS assay of COS7 cells transfected with indicated vectors after siChk1 or nt-siRNA treatment. The ratio of 3H- (after) and 14C- (before) labelled DNA was calculated after 3 J m−2 UV treatment and the relative DNA synthesis is shown. The data are the mean of two experiments using two cell lines from each genotype in triplicates. Error bars represent the s.e.m. (d) Cell viability of COS7 cells stably expressing indicated vectors after depletion of endogenous Chk1 and treatment with 1.25 mM HU for 20 h. Cell numbers relative to untreated controls were quantified by high-content analysis microscopy. The data represent the mean of three independent experiments from each genotype in triplicate. Error bars represent the s.e.m. (e) Flow cytometric analysis of cells treated with 1.25 mM HU for 48 h. The Annexin V- and GFP-double-positive population was plotted. The data represent the mean of two independent experiments from each genotype in duplicate. Error bars represent the s.e.m. (f) Colony formation and dose-response of cells with indicated genotype and treatment. Data shown are one of two experiments. For statistical evaluation, ANOVA followed by Tukey post hoc analysis was performed. **P<0.01; ***P<0.001. NS, not significant.
