Fig. 4. CX-5461 induces R-loops stabilization in HR-proficient OVCAR8 cells.

a Co-immunofluorescence (Co-IF) of GQ DNA and UBF as a nucleolar marker in OVCAR8 cells and OVCAR8 RAD51C KO cells treated with either vehicle, 1 μM CX-5461 or 10 μM TMPyP4 for 3 h. Representative images of three biologically independent experiments. b Quantitation of GQ DNA immunofluorescence. Signal intensities were analyzed using CellProfiler. Error bars represent mean ± SD, n = 200 cells per treatment condition examined over three independent experiments. c Co-IF analysis of R-loops and UBF in cells treated with vehicle or 1  μM CX-5461 for 3 h. Representative images of two biologically independent experiments. d Quantitation of R-loops signal intensity was performed using CellProfiler. n = 230 cells per treatment condition were examined over two independent experiments. Integrated intensity was normalized to corresponding median value of OVCAR8 vehicle control. Statistical analysis in (b) and (d) were performed using a one-sided one-way ANOVA, Kruskal–wallis multiple comparisons test (adjusted p-values are shown). (ns) denotes a non-significant p-value greater than 0.05. e Cells were treated with vehicle, 100 nM or 1 μM CX-5461 for 3 h. RNA was extracted and 47S rRNA precursor levels were determined using primers specific to the 5’ETS of pre-rRNA. Expression levels were normalized to NONO mRNA and expressed as a fold change relative to vehicle (n = 3 biologically independent experiments), error bars represent mean ± SEM, statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p-values are shown).