Fig. 1

JMJD6 is recruited to DSBs, and limits the spreading of histone ubiquitination around DSBs independently of its enzymatic activity. a EGFP-JMJD6 is recruited to DNA damage sites. EGFP-JMJD6 expression constructs were transfected into U2OS cells, and the localization of EGFP-JMJD6 was observed under a fluorescence microscope following laser microirradiation. Scale bar, 20 μm. b The endogenous JMJD6 is recruited to laser irradiated regions. Cell treated with microirradiation were subjected to immunofluorescent staining using anti-JMJD6 together with anti-γH2A.X. Scale bar, 20 μm. c JMJD6 overexpression does not affect the initial γH2A.X foci formation, but prevents the vanishment of those foci. U2OS cells transfected with FLAG-JMJD6 expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-γH2A.X at 1 and 8 h after irradiation, respectively. Scale bar, 20 μm. d JMJD6 overexpression does not alter MDC1 foci formation, but prevents the vanishment of those foci. Cells were transfected with FLAG-JMJD6 expression constructs, exposed to 10 Gy of IR, and immunostained for FLAG and MDC1 at the indicated time. Scale bar, 20 μm. e JMJD6 overexpression inhibits the spreading of histone ubiquitination in response to IR. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with FK2 antibodies. Scale bar, 20 μm. f The spreading of histone ubiquitination in response to IR is increased upon depletion of endogenous JMJD6. U2OS cells transfected with JMJD6 siRNAs or control siRNAs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using FK2 antibodies. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis using anti-JMJD6 and anti-GAPDH. g JMJD6 overexpression inhibits the spreading of RNF168 around DSBs. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-RNF168. Scale bar, 20 μm. h JMJD6 knockdown increases the spreading of RNF168 around DSBs. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis. For Fig. 1c–h, at least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p-value was determined by Student’s t-test. ****p < 0.0001, **p < 0.01, NS = not significant