Fig. 5.

CU1276 modulates proliferation and DNA damage signaling in an RPA1-dependent manner. Growth curves of P3HR1 stable cell lines containing bidirectional, doxycycline-inducible vectors expressing GFP alone (blue line), GFP plus the CU1276 hairpin (red line), or RPA1 plus the CU1276 hairpin (orange line) (A Upper), and corresponding Western blot analysis of RPA1 protein levels from these cell lines, with ACTB used as loading control (A Lower). Growth curve data are compiled from eight independent experiments, with each genotype represented by four independently derived bulk populations. Error bars represent the 95% confidence intervals of each cell type, calculated according to a normal distribution. CU1276 expression is sufficient to significantly reduce cellular proliferation relative to the GFP control at 96 h (Student’s t test, *P = 1.8e-3). At 96 h, RPA1 rescue restores growth completely to wild-type levels. (B) Western blot analysis of RPA1, total H2AFX, and γH2AFX in etoposide-treated control cells and cells expressing CU1276. ACTB was used as loading control. Image is representative of three independent experiments, for which average γH2AFX quantifications are indicated in bar chart format. Error bars represent the SD of three independent experiments. (C) Western blot analysis of control cells, cells expressing CU1276, and cells simultaneously expressing CU1276 and exogenous RPA1. Restoration of RPA1 protein levels rescues CU1276-mediated sensitization of H2AFX phosphorylation upon etoposide treatment. ACTB was used as loading control. Image is representative of three independent experiments, for which average γH2AFX quantifications are indicated in bar chart format. Error bars represent SDs.