Hcs nuclearmask

The oocytes were briefly washed in PBS and then fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After washing in PBS (3× for 10 min), the oocytes were permeabilized for 15 min in 0.1% Triton X 100 in PBS and incubated in 10% fetal bovine serum (FBS) at room temperature. Then, the oocytes were incubated overnight at 4 °C with the primary anti-γ-H2AX antibody (Cell Signaling Technology, Danvers, MA, USA, 9718) diluted 1 : 100 in 10% FBS. After washing in PBS (3× for 10 min), the oocytes were incubated for 1 h at room temperature with secondary Cy3-conjugated goat anti-rabbit antibody (Abcam, Cambridge, UK, ab6939), used at a dilution of 1 : 500 in 10% FBS. For nuclear DNA visualization, after washing in PBS (3× for 10 min), the oocytes were incubated for 15 min in HCS NuclearMask™ (Thermo Fisher Scientific), then washed in PBS for 10 minutes, and mounted in 1 μl PBS droplets using a Vaseline layer between a slide and a cover glass to preserve the oocyte three-dimensional structure. The mounted specimens were analyzed with the ZEISS LSM 880 Confocal Laser Scanning Microscope (Oberkochen, Germany) using the 20× Zeiss Plan-Apochromat Infinitive corrected objective with a numerical aperture of 0.8. Fluorescence signal intensities from irradiated oocytes were measured using ImageJ software and normalized to control (non-irradiated oocytes).

CHO cells were seeded onto glass bottom, 96 well plates and transfected with RUSH-APOL1 plasmids. 24 hr after transfection, cells were treated with or without 80 µM biotin every 30 min for 0–120 min. For cell surface immunostaining, cells were moved onto ice and blocked with HBSS + Ca²⁺ + Mg²⁺ + 0.5% BSA fraction V, stained with primary antibodies, fixed in 2% formaldehyde (Thermo 28906), quenched with 50 mM NH₄Cl, and then stained with secondary antibodies. For intracellular staining, cells were permeabilized with 0.075% saponin. Staining was performed with the following antibodies and dyes: mouse anti-APOL1 1:800, rabbit anti-calnexin 1:200 (Stressgen, Farmingdale, NY. SPA-860), anti-rabbit Alexa 488 plus 1:1500 (Thermo A32731), anti-mouse Alexa 647 1:1000 (Thermo A21236), and HCS Nuclear Mask 1:400 (Thermo H10325). Cells were imaged via spinning disk confocal microscopy.