Figure 4.

Reduced homologous recombination (HR) efficiency provoked by AR suppression treatment results from cell-cycle alterations. (A) LNCaP, PC346C, and PC346C-Flu1 cells were treated with AR suppression for 48 h and RAD51 expression was analyzed by Western blotting. Tubulin was used as a loading control. Full blots can be found in Figure A3 (Appendix). (B) Quantification of RAD51 protein levels compared to loading control and normalized to untreated (Ctrl). Averages and SEM are indicated. (C) The same cell samples from a were incubated with EdU for 30 min before fixation and the cell-cycle profile was determined by flow cytometry. Averages and SD are indicated. (D) A transient directed repeats-green fluorescent protein (DR-GFP) assay was performed in LNCaP and PC346C-Flu1 cells treated with apalutamide for 48 h. Cells transfected with small interfering RNA (siRNA) against breast cancer 1 (BRCA1) transcript for 72 h were used as a positive control. GFP-positive cells were scored by flow cytometry and quantified. Averages and SD are indicated. (E) Representative images of double staining of EdU-positive and RAD51 foci and their colocalization in LNCaP cells at 2 h after 5 Gy of IR treatment (scale bar = 10 μm). (F) Quantification of RAD51 foci numbers in EdU-positive cells. Each data point represents one cell; averages and SEM are indicated; * p < 0.05, ** p < 0.01, ns, non-significant. ADT, androgen-deprivation treatment; APA, apalutamide; ENZA, enzalutamide.