Dm 6000rxa fluorescence microscope

Cells were grown on coverslips and treated with or without dox (1 µg per mL) for indicated time points. For RAD51 foci formation, cells were irradiated with 5 Gy using a CIS international/IBL 637 cesium¹³⁷ source. After 3 h of irradiation, cells were washed with PBS and fixed in 2% formaldehyde with 0.1% Triton X-100 in PBS for 30 min at room temperature. Cells were permeabilized in 0.5% Triton X-100 in PBS for 10 min and subsequently blocked with PBS containing 0.05% Tween-20 and 4% BSA for 1 h. For micronuclei staining, cells were fixed in 4% formaldehyde for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS for 1 min followed by blocking in 0.05% Tween-20 and 2.5% BSA in PBS for 1 h. Cells were incubated overnight with primary antibodies against RAD51 (1:400, GeneTex, #gtx70230), Geminin (Cell Signaling, #9718, 1:200) or cGAS (1:200, Cell Signaling, #15102) in PBS–Tween–BSA. Cells were extensively washed and incubated for 1 h with Alexa-conjugated secondary antibodies (1:400) at room temperature in the dark. Slides were mounted with ProLong Diamond Antifade Mountant with DAPI (Thermo Scientific). Images were acquired on a Leica DM-6000RXA fluorescence microscope, equipped with Leica Application Suite software.

To assess replication fork protection during replication stress, KBM-7-shBRCA2 #2 cells were pre-treated with doxycycline (1 μg per mL) for 96 h, and then pulse-labeled with chloro-deoxyuridine (CIdU, 50 µM) for 40 min. Subsequently, cells were washed with medium and incubated with HU (2 mM) for 4 h. Cells were lysed on microscopy slides in lysis buffer (0.5% sodium dodecyl sulfate (SDS), 200 mM Tris (pH 7.4), 50 mM EDTA). DNA fibers were spread by tilting the slide and were subsequently air-dried and fixed in methanol/acetic acid (3:1) for 10 min. Fixed DNA spreads were stored for 24 h at 4 °C, and prior to immuno-labeling, spreads were treated with 2.5 M HCl for 1.5 h. CIdU was stained with rat anti-BrdU (1:750, AbD Serotec) for 2 h and slides were further incubated with AlexaFluor 488-conjugated anti-rat IgG (1:500) for 1.5 h. Images were acquired on a Leica DM-6000RXA fluorescence microscope, equipped with Leica Application Suite software. The lengths of CIdU and IdU tracks were measured using ImageJ software.