Fig. 7
The impaired DDR mediated by JMJD6 overexpression is SIRT1- and BRD4 dependent. a SIRT1 knockdown counteracts the impaired 53BP1 foci formation in JMJD6-overexpressed cells. U2OS cells stably expressing shRNAs specific for SIRT1 or control shRNAs were transfected with FLAG-JMJD6 expression constructs, and immunofluorescence experiments were performed using anti-FLAG together with anti-53BP1 1 h after IR treatment. Scale bar, 20 μm. 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. b Overexpression of FLAG-JMJD6-N inhibits the recruitment of 53BP1 to DSBs. U2OS cells transfected with FLAG-JMJD6-N expression constructs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with anti-53BP1. Scale bar, 20 μm. *p < 0.05. c BRD4 is essential for the recruitment of JMJD6 to chromatin. U2OS cells transfected with control or BRD4 specific siRNAs were treated with or without IR. Chromatin-bound proteins or total proteins were extracted and then subjected to western blot analysis using indicated antibodies. d The impaired loading of 53BP1 in JMJD6-overexpressed cells is counteracted by BRD4 inhibition. U2OS cells transfected with FLAG-JMJD6 expression constructs were untreated or treated with JQ1, and 1 h after IR treatment, immunofluorescence experiments were performed using anti-FLAG together with anti-53BP1. Scale bar, 20 μm. 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
