4 hydroxytamoxifen

In the FOK1 system, DSBs were induced at the LacO array by inducing the nuclear localization [4-hydroxytamoxifen (100 nM, #3412, Tocris)] and stabilization (Shield-1 ligand, 0.5 μM; CIP-S1-0001, CheminPharma) of the ER-mCherry-LacR-FOK1-DD nuclease for 4 hours before immunofluorescence sample preparation. Replication fork stall at the LacO array was achieved by transfecting the empty mCherry-LacR fusion protein 24 hours before fixation. DNA damages were induced by exposing cells to either ionizing irradiation (IR 1 or 10 Gy) with a CellRad (Precision X-Ray Inc.), cisplatin treatments (Tocris; 1 μM for 3 or 24 hours at 250 nM in time course experiment and 2 μM in survival assay), MAF (1.30 and 1.57 μM in Namalwa and Raji screens, respectively; Toronto Research Chemicals), HU (4 and 1 mM in survival assay; Sigma-Aldrich), formaldehyde (150 μM; Sigma-Aldrich), MMC (150 nM; Sigma-Aldrich), talazoparib (3 μM; BMN 673, Selleck Chemicals), or UV (10 J/m²). UV irradiations were carried on with a germicidal lamp (243 nm).

Cells were treated with 10 nm 17β-estradiol (Sigma-Aldrich, St. Louis, MO, USA), 500 nm 4-hydroxytamoxifen (Tocris Bioscience, Bristol, UK), ethanol, or with DMSO as a vehicle. Cell proliferation was measured in one of two ways. Trypan blue exclusion assays were used to manually count cells using a hemocytometer. Otherwise, cell proliferation was measured using a standard MTS reagent, CellTiter96 Aqueous One Solution (Promega, Madison, WI, USA), according to the manufacture’s standard protocol. For combination treatment experiments, 7500 MCF-7 or T47D cells were seeded in a 96-well format, whereas 5000 MDA-MB-231 cells were similarly seeded for experimentation. Statistical analysis of these experiments was carried out using a standard two-tailed Student’s t-test. All experiments were performed in triplicate. BRAF knockdown was accomplished by transfecting breast cancer cell lines with one of two targeting siRNAs (BRAF siRNA 1: J-003460-12-0005, BRAF siRNA 2: J-003460-13-0005) following the standard manufacturer’s protocol (Thermo Scientific Dharmacon, Lafayette, CO, USA). Scrambled siRNA from the same manufacturer were utilized as negative controls. In these experiments, 5000 MCF-7 cells were seeded into a 96-well format for knockdown and subsequent MTS assays.