Mouse anti phospho histone h2a x ser139

For γH2AX and RAD51 immunofluorescence, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with 1% normal goat serum (for γH2AX) or 3% BSA (for RAD51) in PBS for 1 h. For TOP1cc staining, cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, permeabilized with 0.25% Triton X-100 in PBS for 15 min at 4 °C and treated with 1% SDS in PBS for 5 min at room temperature. Cells were washed five times with wash buffer (0.1% Triton X-100, 0.1% BSA in PBS) and blocked with 10% milk in 10 mM Tris-HCl pH 7.4 and 150 mM NaCl. Primary antibodies used were mouse anti-phospho-Histone H2A.X (Ser139) (Millipore, 05-636, 1:500), rabbit anti-RAD51 (Calbiochem, PC130, 1:300), and mouse anti-TOP1cc antibody^{53 (link)} (1:100). Secondary antibodies used were goat anti-mouse IgG Alexa fluor 488 (Invitrogen, A11029, 1:2000) and goat anti-rabbit IgG Alexa fluor 488 (Invitrogen, A11034, 1:2000). Images were captured using a Zeiss Axio Scope.A1 fluorescent microscope equipped with a CCD camera (Jenoptik). Foci were scored using ImageJ on images captured with a 63× objective (for γH2AX) or counted manually (for RAD51 and TOP1cc). At least 100 cells were scored for focus formation by blinded observers.

After 27HC or control treatment of LNCaP, DU45 and RWPE-1, cells were fixed with 10% trichloroacetic acid for 30 min on ice, and the total cell lysates were prepared with LDS Sample Buffer (Thermo Fisher Scientific). Western blotting was performed with NuPAGE protein electrophoresis system (Thermo Fisher Scientific). Primary antibodies included rabbit anti-Rad51 (1:500, sc-8349, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-FEN1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-Histone H2A.X (Ser139, 1:1000, 05-636-I, Millipore Sigma, St. Louis MO, USA), rabbit anti-phospho-KAP1 (1:1000, Bethyl Laboratories, Thermo Fisher Scientific), rabbit anti-phospho-RPA32 (1:1000, Bethyl Laboratories Thermo Fisher Scientific) and mouse β-actin (1:1000, Santa Cruz Biotechnology). Ponceau S staining solution (Thermo Fisher Scientific) was used to evaluate transfer efficiency and for total protein normalization. Bands were visualized with Immun-Star AP Chemiluminescence Kits (Bio-Rad Laboratories) and were detected with ChemiDoc Touch Imaging System (Bio-Rad Laboratories) and normalized to loading controls (β-actin/Ponceau S) first and then to each control group (0 µM). Results were run in triplicate.