Immunofluorescence and Confocal Microscopy Protocol

Immunofluorescence staining and confocal microscopy were performed as described previously (10 (link),18 (link),19 (link)). Briefly, cells were grown and drug treated on chamber slides (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber slides) followed by fixation with 4% paraformaldehyde for 10 min at room temperature. Primary antibodies against PRMT5, γH2AX and XRCC1 were detected using anti-rabbit or anti-mouse IgG secondary antibodies labeled with Alexa 488/568 (Invitrogen). Cells were mounted in anti-fade solution with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and examined under Leica TCS SP8 confocal laser-scanning microscope (Germany) with a 63×/1.4 NA oil objective. Images were collected and processed using the Leica software and sized in Adobe Photoshop 7.0. The γH2AX intensity per nucleus was determined with Adobe Photoshop 7.0 by measuring the fluorescence intensities normalized to the number of cell count (10 (link),18 (link),19 (link)).