Figure 5.
Inhibition of PLK1 induces mitotic failure and apoptosis in the FANCA-deficient hTERT-RPE1 cell line. A, Representative Western blot showing FANCA expression in control (Scr.Ctrl.) cells vs. FANCA-KD (shFANCA) hTERT-RPE1 cells after 48 hours of doxycycline (Dox) treatment. B, Concentration-dependent survival (%) of shFANCA and Scr.Ctrl cells treated with volasertib and onvansertib for 3 days. Cell viability was measured by CellTiter-Glo assay. Representative graph shows the mean ± SEM cell survival (%) compared with vehicle. P values were calculated by two‐way ANOVA. C, Concentration-dependent cellular apoptosis (fold change) of shFANCA and Scr.Ctrl. hTERT-RPE1 cells treated with volasertib for 3 days. Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay. Representative graph shows the mean ± SEM caspase 3/7 activity (fold change) compared with vehicle. P values were calculated by two‐way ANOVA. D, Experimental design and a representative Western blot showing increased cyclin B1 and γH2AX in shFANCA vs. Scr.Ctrl. hTERT-RPE1 cells. E, Experimental design and representative live-cell imaging images of shFANCA and Scr.Ctrl. hTERT-RPE1 cells stably expressing mCherry–tubulin (red) and GFP–H2B (green) after DMSO and volasertib treatment. Scale bars in the image panels represent 0.03 μm. Representative graph showing quantification of cells undergoing mitotic collapse and cytokinesis failure (n = 50 cells per group). Graph shows the mean ± SEM. P values were calculated by one‐way ANOVA. F, Representative IF images show γH2AX foci (red) and UFBs (RPA70, green) in shFANCA and Scr.Ctrl. mitotic hTERT-RPE1 cells after vehicle and volasertib (20 nmol/L) treatment. Scale bars in the image panels represent 2 μm. Representative graph showing quantification of γH2AX foci (n ≥ 10 cells per group) in DMSO-treated metaphase (+) cells vs. volasertib-treated (20 nmol/L) metaphase-collapsed (#) cells. Graph shows the mean ± SEM. P values were calculated by one‐way ANOVA. All graphs are representatives of at least three independent experiments.