Skip to main content
. 2021 Oct 1;12:5779. doi: 10.1038/s41467-021-26049-6

Fig. 5. Prostate cancer-derived SPOP mutants increase replication origin firing, re-replication, and genome instability, especially upon ATR inhibition.

Fig. 5

a PC-3 cells stably expressing empty vector (EV) and SPOP-F133V mutant were synchronized by thymidine and L-mimosine double blockade. Cells were released and harvested at the indicated time points for FACS-based cell cycle analysis. b PC-3 cells expressing EV and SPOP F133V were synchronized as in (a) and harvested for WB. Similar results were obtained in three independent experiments. c Protein bands for chromatin-associated MCM2 from the experiment shown in (b) and the other two repeats were quantified using ImageJ and normalized to total MCM2 protein level. Data are presented as the mean ± SD (n = 3). The P value was calculated by two-way ANOVA analysis. d EV- and F133V-expressing PC-3 cells transfected with HA-Ub-K27-only were synchronized with nocodazole and released at different time points and harvested for IP and WB. e, f PC-3 cells infected with lentivirus expressing EV or SPOP F133V in combination with control or Geminin-specific shRNAs. Cells were pulse-labeled with 30 μM BrdU for 30 min and harvested for FACS analysis (e) and quantitative data are shown in (f). Data are shown as the mean ± SD of three independent experiments. Two-tailed unpaired Student’s t-test. g PC-3 cells infected with lentivirus expressing empty vector (EV) or F133V in combination with control or Geminin-specific shRNAs were treated with DMSO or 100 nM VE-822 for 8 h followed by DNA fiber assay. Inter-origin distances between 200 replication firing origins were measured (n = 200). Data are shown as the mean ± SD of three independent experiments. Two-tailed unpaired Student’s t-test. h Stable PC-3 cells as indicated were treated with DMSO or 100 nM VE-822 for 8 h followed by DNA fiber assay. DNA re-replication was quantified from 200 DNA tracks and relative re-replication fold changes are presented. Data are shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. i PC-3 cells infected with lentivirus expressing the indicated shRNAs or EV or SPOP F133V were treated with DMSO or increased doses of VE-822 for 24 h and harvested for WB with indicated antibodies. j Stable PC-3 cells as indicated were treated with VE-822 (100 nM) for 24 h. Cells were harvested for karyotyping. Quantification of chromosome breaks per cell are shown. More than 70 metaphases from four biological replicates were counted. Data are mean ± SEM. Two-tailed unpaired Student’s t-test. Source data are provided in this paper or Mendeley database (10.17632/8n7xt5rkhc.1). Similar results for (b), (d), and (i) panels were obtained in two independent experiments.