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. 2016 Jul 20;7:12235. doi: 10.1038/ncomms12235

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(a) Nuclei from U2OS cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were incubated with 40 gel units of MNase for 5 min followed by DNA extraction and ethidium bromide staining (left). The band densities were quantified using ImageJ software and expressed as percentage of signal minus background of the entire line from top to the bottom. Calibrated kilobase pair (kbp) sizes are indicated (right). (b) U2OS cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were extracted in lysate buffer containing 1.5 M NaCl, salt soluble proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and H3 was detected by western bloting. Ponceau S staining indicates loading. The efficiency of overexpression of FLAG-H3, H3 mutants or total H3 was monitored by western blotting of whole-cell lysate, with corresponding antibodies. (c) Control or SIRT7-depleted U2OS cells were exposed to 10 Gy of IR and collected at different time points. Nuclei were prepared and subjected to MNase assays. Mononucleosome, dinucleosome and trinucleosome are indicated (upper). The band densities were quantified using ImageJ software and the intensity values were background subtracted (lower). (d) Control or SIRT7-depleted U2OS cells were exposed to 10 Gy of IR and collected at different time points. Cells were extracted in lysate buffer containing 1.0 M NaCl, salt-soluble proteins were separated by SDS–PAGE, and γH2AX, H2AX and H3 were detected by western bloting. Ponceau S staining indicated loading. (e,f) Overexpression of H3K122 mutants affected the repair efficiency of NHEJ and HR. EJ5-GFP-HEK293 (e) or DR-GFP-U2OS (f) cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were transfected with I-SceI for 48 h and analysed by FACS. Each bar represents the mean±s.d. for triplicate experiments. **P<0.01 (two-tailed unpaired Student's t-test). The efficiency of overexpression of FLAG-H3, H3 mutants or HA-I-SceI was monitored by western blotting.