Geltrex coated cover slips

MCF7 cells were plated on poly-l-lysine-coated cover slips (Fisher, Hampton, NH), while T47D, MDA-MB-231, and MDA-MB-468 cells were plated on Geltrex^®-coated cover slips (Invitrogen, Carlsbad, CA). Following treatment, the cells were fixed in 1% PFA for 10 min, washed twice with PBS, subsequently permeabilized in 0.3% NP-40/PBS for 10 min, and blocked in Image-iT FX signal enhancer solution (Invitrogen, Carlsbad, CA) for 30 min. Cells were incubated with ERα (1:50 dilution, SC-8005 Santa Cruz Biotechnology, Dallas, TX) and raptor (1:400, ab169506 Abcam, Cambridge, UK) primary antibodies in 1% BSA/PBS overnight at 4 °C. Cover slips were subsequently washed in PBS and incubated with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 goat anti-rabbit secondary antibodies (1:500 dilution, Invitrogen, Carlsbad, CA) for 1 h at room temperature in the dark. Following 5-min incubation with DAPI, cover slips were mounted using an Image-iT^® FX signal enhancer (Invitrogen, Carlsbad, CA) and imaged using a Nikon fluorescent microscope under ×40 magnification.

For immunofluorescence staining, cells grown on geltrex-coated coverslips (Invitrogen, Carlsbad, CA, USA, cat.no. 14113-202) and fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MI, USA, cat.no. P6148), permeabilized using 0.25% Triton-X (Sigma-Aldrich, St. Louis, MI, USA, cat.no. T9284) followed by blocking for 30 min in 10% Bovine Serum Albumin (BSA) (Bovine Albumin Fraction V, 7.5% solution; Thermo Fisher Scientific, Waltham, MA, USA, cat.no. 15260037). Cells were incubated with respective primary antibodies (H2AX (Ser139) (Merck Millipore, Burlington, MA, USA, cat.no. 05-636); Cyclin A (H-432) (Santa Cruz Biotechnologies, Dallas, TX, USA, cat.no. sc-751) in 2% BSA (Bovine Albumin Fraction V, 7.5% solution; Thermo Fisher Scientific, Waltham, Massachusetts, cat.no. 15260037) for 2 h followed by incubation with appropriate secondary conjugated antibodies (Alexa Flour 488 (Invitrogen, Carlsbad, CA, USA, cat.no. A11029); Alexa Flour 568 (Invitrogen, Carlsbad, CA, USA, cat.no. A11036). Nuclei were counterstained with 4,6-Diamidino-2-Phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MI, USA, cat.no. D9542-10MG). Automated imaging and analysis were performed using Olympus Scan-R screening station equipped with Scan-R analysis software as described previously [29 (link)].