Model lsm880 confocal microscope

Cells were grown on coverslips before treatment. Cells were washed in PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% TritonX-100 in PBS for 3 min, and blocked in 2% BSA in PBS for 30 min. Primary antibodies -TOM20 (sc-17764, Santa Cruz), PHB2 (ab182139, abcam), LC3 (14600–1-AP, Proteintech, Manchester, UK)- were used at 1:200 overnight at 4 °C, followed by secondary antibodies at 1:1000 and DAPI (0.5 µg/ml) for 1 h at room temperature. Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647 were obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Cells were mounted with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Images were acquired with a Carl Zeiss model LSM880 confocal microscope and a Apochromat 63×/1.4 Oil M27 objective lens and they were collected using 405, 488, and 561 nm laser lines for excitation and appropriate emission filters. Images were analyzed with Image J Software. Pearson’s coefficient was obtained with JACoP plugin [26 (link)].

FokI-U2OS cells, a stable cell line with a FokI restriction enzyme site, were plated in a four-well plate and transfected with control, MSH2, or MSH3 siRNA. The next day, Lipofectamine 3000 was used to transfect the cells with LacI-mCherry-FokI expression plasmid and GFP-EXO1 or mNeon-MRE11. After 48 h, the transfected cells were stained with Hoechst for 15 min to visualize the nuclei. Live cell images were obtained using a model LSM880 confocal microscope (Carl Zeiss).