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. 2018 Mar 23;9(22):15915–15930. doi: 10.18632/oncotarget.24636

Figure 3. KDM2A modulates the recruitment of 53BP1 to DSBs and entry into mitosis upon IR.

Figure 3

(A) KDM2A modulates 53BP1 accumulation at DSBs generated by irradiation. U2OS cells transfected with control siRNA (siCTL) or siRNA targeting the 3’-UTR region of the KDM2A mRNA (siUTR) and stably expressing MYC-tagged KDM2A variants were irradiated (2Gy), fixed 1h later and subjected to immunofluorescence analysis with DAPI, anti-53BP1 (green) and anti-MYC (red). Quantification of immunofluorescence analyses (right panel) shows the average number of 53BP1 IRIF per cell in each condition. Experiments were performed in triplicate. Single (*), double (**) and triple (***) asterisks indicate p<0.05, two-tailed paired Student t-test. Error bars indicate standard deviation. (B) KDM2A depletion impairs 53BP1 recruitment to DNA breaks generated by FokI endonuclease. U2OS-DSB-reporter cells transfected with siCTL or siKDM2A were treated with Shield-1 and 4-OHT to promote expression of mCherry-LacI-FokI (FokI) (red). Immunofluorescence analyses were conducted with anti-53BP1(green) and anti-KDM2A. Quantification of immunofluorescence analyses (right panel) shows the percentage of cells displaying colocalization between 53BP1 and FokI. Asterisk indicates p<0.05, two-tailed paired Student t-test. Error bars indicate standard deviation. (C) KDM2A depletion results in faster progression into mitosis without proper repair of damaged DNA. U2OS cells transfected with siCTL or siKDM2A were left untreated or irradiated (2Gy) and harvested at the indicated time points after irradiation. The percentage of mitotic cells, as indicated by H3pSer10 staining, was determined by flow cytometry. Data shown is representative of experiments replicated three times.