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. 2016 Dec 7;8(2):2916–2935. doi: 10.18632/oncotarget.13807

Figure 3. The SUMOylation increase of HIC1 upon induction of irreparable DSBs is dependent on ATM but independent of DNA-PKcs.

Figure 3

(A) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), either with a pool of four siRNAs targeting ATM (siATM) or with a pool of four siRNAs targeting DNAPKcs (siDNAPKcs). The next day, these cells were transfected with a FLAG-HIC1 expression vector for 24 hours and were then treated with 20 μM etoposide (+) or mock-treated with DMSO (–) as control for 16 hours before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. (B) Quantification of SUMO-HIC1. The HIC1 SUMOylated band in control conditions (siCtrl, DMSO 16 h; lane 1 in panel A) was quantified with the Fujifilm MultiGauge software and given the arbitrary value of 1. The other HIC1 SUMOylated bands (lanes 2 to 6 in panel A) were quantified relative to this value. (C) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), a pool of four siRNAs targeting ATM (siATM) or with each individual siRNA from the pool targeting DNA-PKcs (siDNA-PKcs). Then, cells were treated with etoposide and total cell extracts were analyzed by Western blot on three different gels (two 6% polyacrylamide gels for DNAPK-cs and ATM; a 15% polyacrylamide gel for γH2AX, H2AX and actin) as described in panel A.