Fig. 4

USP48 antagonizes BRCA1-mediated resection. a Increased resection seen on USP48 knockdown requires BRCA1 and SMARCAD1. Resection lengths were measured in HeLa cells depleted as indicated. Cells were treated with 10 mM BrdU for 24 h with addition of 10 µM Olaparib for the final 16 h. Cells were lysed and DNA fibres spread before staining for BrdU-positive single-stranded DNA resection tracks. n = 190 tracks for each condition. Bars indicate median, also shown numerically in brackets. ***p < 0.005 Mann–Whitney test. b Western blottings to demonstrate protein expression levels in HeLa cells following siRNA as indicated. c Rad51 foci formation in S-phase (EdU positive) HeLa cells depleted as indicated. Cells were fixed at 2 h post 5 Gy IR. Scale bars = 10 µm (see Supplementary Fig. 6a, b for quantification). d Suppressive impact of USP48^Iso2 overexpression on resection lengths can be rescued by co-expression of an uncleavable H2A-Ub fusion. Resection lengths were measured in untransfected HeLa cells or those transfected with USP48^Iso2 and expressing either H2A or H2A-Ub fusion (KR2 denotes that lysines 13/15, 118/119, and 125/127/129 were mutated to arginines). Cells were prepared as in a and 210 tracks were measured for each condition. Bars indicate median, also shown numerically in brackets. ***p < 0.005, NS = nonsignificant, Mann–Whitney test. e USP48 restricts positioning of 53BP1 in damage foci. Images of BRCA1 and 53BP1 in cells treated with control or USP48 siRNA exposed to 2 Gy IR and fixed 8 h later. Scale bars = 2 µm. Quantification of mean relative intensity profiles for co-localizing foci. n = 25, bars = SEM. Right panel shows western blotting of USP48 protein levels