Fig. 1

USP21 promotes HR by facilitating RAD51 accumulation at DSBs. a HR efficiency in TRI-DRGFP reporter cells in the presence of the indicated shRNAs or transient USP21 overexpression. One of at least three representative experiments is shown. b HR efficiency in control (WT) TRI-DRGFP cells and two independent CRISPR/Cas9 USP21 knockout (KO) clones. c NHEJ efficiency in pEJ5-GFP-transgenic U2OS cells transfected with the indicated siRNAs. Values in a through c are expressed as mean and s.d. and samples were analyzed in triplicate. d–h Immunofluorescence (IF) analyses at laser-induced DSBs in U2OS cells 1 h after release from double-thymidine-block. Representative images are shown, scale bar: 20 µm. For box plots, bottom and top hinges denote first and third quartile, respectively. Whiskers denote ±1.5 interquartile range (IQR). The red horizontal line represents the median. p-values are based on a two-sided Wilcoxon rank-sum test, sh-U21: sh-USP21. d γ-H2AX levels in cells expressing sh-Luc (n = 30), sh-U21-1 (n = 26) or sh-U21-2 (n = 35). One of five representative experiments is shown. Fluorescence intensity of γ-H2AX⁺ areas was quantified and normalized to sh-Luc. e CtIP accumulation in cells expressing sh-Luc (n = 28), sh-U21-1 (n = 27) or sh-U21-2 (n = 36). Fold enrichment reflects the ratio of γ-H2AX⁺ over γ-H2AX^– nuclear areas. f RPA accumulation at laser-induced DSBs as in e; sh-Luc (n = 23), sh-U21-1 (n = 22), sh-U21-2 (n = 23). g RAD51 accumulation at laser-induced DSBs as in e. One of five representative experiments is shown, sh-Luc (n = 30), sh-U21-1 (n = 32), sh-U21-2 (n = 37). h RAD51 accumulation as in g using control (WT, n = 60) and USP21 KO cells (n = 55). i IF analysis of RAD51 foci 6 h after IR (10 Gy). Box plots represent the number of nuclear RAD51 foci (green); sh-Luc (n = 284), sh-U21-1 (n = 145), sh-U21-2 (n = 174). Representative analyses and images are shown for one of two independent experiments, scale bar: 10 µm. Unless noted otherwise, p-values are based on Student’s two-tailed t-test: *p < 0.05, **p < 0.01, ***p < 0.001