Fig. 3

USP48 deubiquitinase activity restrains resection. a USP48 is recruited to sites of damage. BrdU-sensitized HeLa cells were subjected to laser stripe IR followed by fixation after 30 min and staining for endogenous USP48, BRCA1, and 53BP1. Scale bar = 10 µm. In the middle and lower panel, cells were treated with USP48 (exon 5 and exon 11 targeting sequences) and BRCA1 siRNA, respectively. USP48 siRNA treatment utilized exon 5 and exon 11 targeting sequences together throughout the manuscript, unless otherwise stated. b Percentage of USP48-positive laser lines in control vs BRCA1-depleted cells. Graph is mean from three independent experiments. Error bars are SEM and *p < 0.05 Student’s T-test. c, d Expression levels of USP48 in stable HeLa-FlpIn cell lines following depletion of USP48 and DOX-induced expression of siResistant EGFP-USP48^Iso1 c or Flag-USP48^Iso2-WT or Flag-USP48^Iso2-C98S d. e, f USP48 depletion increases resection lengths. Resection lengths were measured in HeLa cells depleted for USP48 and complemented as in c and d. Cells were incubated 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. One hundred and twenty fibres were measured using ImageJ for each condition. Bars indicate median, also shown numerically in brackets. ***p < 0.005 Mann–Whitney test