Fig. 3

Impact of BRPF1 pharmacological blockade on metastatic potential, cell cycle, death, and DNA damage response of OC cells. (A) Relative cell viability was assessed in cells treated with indicated GSK6853 concentrations at displayed time points, determined by MTT assay in OVCAR-3 (left) and PEO4 (right) cells. Vehicle (DMSO) was used as control. Data represent the mean of six independent replicates ± SD (* p < 0.05). (B) Wound healing assay in OVCAR-3 and PEO4 after treatment with 10 µM GSK6853 or DMSO (CONTROL) as negative control for 12 days. Quantitative analysis of scratch width was performed at 0 and 24 h time points. Data are expressed as mean ± S.D. of five independent replicates (Supplementary Fig. S5). Stars indicate statistically significant differences (****p < 0.0001). (C) Colony formation assays were performed in OVCAR-3 (upper panel) and PEO4 (lower panel) cells after treatment with 10 µM GSK6853 or DMSO as control. Numbers displayed below images of cell culture plates indicate the number of colonies present in the corresponding well. The results reported on histograms represent an average of three independent measurements and are analyzed with respect to the DMSO (CONTROL). Stars indicate statistically significant differences (*p < 0.005, ****p < 0.0001). (D) Histograms displaying the results of cell invasion assay performed in OVCAR-3 (left panel) and PEO4 (right panel) grown in the presence of 10 µM GSK6853 or DMSO as control (CONTROL). The results reported on histograms represent the average of independent measurements and are analyzed with respect to the DMSO (CONTROL). Stars indicate statistically significant differences (*p < 0.05; ***p < 0.0001). (E) Histograms showing the results of cell cycle analysis performed after 12 days of GSK6853 (10 and 20 µM) treatment or DMSO as a control in OVCAR-3 (left panel) and PEO4 (right panel) cells. The reported results represent the average of three independent measurements. Stars indicate statistically significant differences (*p < 0.05; **p < 0.001). (F) Histograms displaying the results of the Caspase 3/7 activity assay following BRPF1 blockade with 10 µM GSK6853 in OVCAR-3 and PEO4 cells for 12 days. Data are presented as the mean ± S.D. of determinations from a representative experiment performed in six independent replicates after 12 days of treatment. All data are analyzed with respect to the DMSO (CONTROL). Asterisks indicate statistically significant differences (*p < 0.05, ****p < 0.0001). (G) Representative western blot showing cleaved Caspase 3, γH2A.X, pH2A.X (Tyr142) protein levels following 10 µM GSK6853 treatment for 12 days in OVCAR-3 and PEO4 OC cells. β-Actin and total Histone H3 were used as loading controls. (H) Representative western blot showing PARP, RAD51, Bcl-2, and p21 protein levels following 10 µM GSK6853 treatment for 12 days in OVCAR-3 and PEO4 OC cells. β-Actin was used as a loading control. The uncropped blots are presented in Supplementary Fig. S1.