Sc 271682

Cells were grown on sterile 12 mm glass coverslips, fixed in 3% formaldehyde in PBS for 15 min at room temperature, washed once in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.2% Triton X-100 (Sigma-Aldrich), and washed twice in PBS. All primary and secondary antibodies (Alexa fluorophores; Life Technologies) were diluted in filtered DMEM containing 10% FBS and 0.02% Sodium Azide. Antibody incubations were performed for 2 h at room temperature. After antibody incubations, coverslips were washed once with PBS and incubated for 10 min with PBS containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) at room temperature to stain DNA. After three washing steps in PBS, coverslips were briefly washed with distilled water and mounted on 6 μl Mowiol-based mounting media. The following primary antibodies were used for immunostaining: H2AX Phospho S139 (mouse, 613401, 1:1,000; BioLegend), 53BP1 (mouse, Upstate MAB3802, 1:1,000), H4K20me2 (rabbit, ab9052, 1:100; Abcam), H4K20me1 (rabbit, ab9051, 1:200; Abcam), BRCA1 (mouse, sc-6954, 1:100; Santa Cruz), Cyclin A (mouse, sc-271682, 1:100; Santa Cruz), and RAD51 (rabbit, 70-002, 1:1,000; Bioacademia).

Cells were plated, allowed to adhere overnight, and pretreated with tamoxifen or fulvestrant one hour prior to irradiation. Cells were harvested at indicated time points and lysed using RIPA buffer (ThermoFisher 89901) containing protease and phosphatase inhibitors (Sigma-Aldrich PHOSS-RO, CO-RO; Santa Cruz Biotechnology sc-3540, sc-24988A; Cayman Chemical 14333, 14405). Samples were separated on precast NuPAGE Bis-Tris protein gels (ThermoFisher), transferred to PDVF membranes (Millipore IPVH00010), and blocked with blotting grade blocker (BioRad 1706404, 5% milk). Antibodies used for protein detection include ERα (Cell Signaling 8644S; 1:1000), Rad51 (Millipore PC130; 1:500), β-actin (Cell Signaling 12262S; 1:50,000), total PARP1 (Abcam ab6079; 1:1000), cleaved PARP (Cell Signaling 5625S; 1:1000), cyclin E1 (Santa Cruz sc-247; 1:1000), cyclin B (Santa Cruz sc-245; 1:1000), and cyclin A (Santa Cruz sc-271682; 1:1000). Secondary antibodies used include anti-mouse (Cell Signaling 7076S; 1:10,000) and anti-rabbit (Cell Signaling 7074S; 1:10,000). All blots are derived from single experiments and are processed in parallel. Quantification of western blots was performed with ImageJ software.

Antibodies against VEGFR2 (#2479), cyclin D1 (#2978S), cyclin B1 (#4135S), FAK (#3285, 1:1,000), phosphorylated (p)-FAK (Y397; #3283,), β1 integrin (#9699,), Akt (#9272,), p-Akt (S473; #9271, 1:1,000), caspase 3 (#9662S), cleaved PARP (#9541), p44/42 MAPK (ERK1/2; #9107), p-p44/42 MAPK (Thr 202/Tyr 204; #9101), vimentin (#3390), N-cadherin (#5296), β-catenin (#8480), GAPDH (#2118) and E-cadherin (#3195) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cyclin A (sc-271682) and FAK (sc-558) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Cisplatin was purchased from Sigma (St. Louis, MO, USA).