Fig. 6

TRIM28 positively regulates the protein stablity of SETDB1. A IF-FISH of TRIM28 KD U2OS cells was performed using a telomere probe (green) and antibodies against SETDB1 (red). Arrows indicate co-stained signals. B 200 cells were analyzed for each line and those with ≥ 5 SETDB1-telomere co-localized foci were counted as positive. Error bars represent SD (n = 3). ***P < 0.001, Student’s t-test. C Cells from (A) were lysed for western blot analysis with the indicated antibodies. D TRIM28 overexpressing U2OS cells were treated with cycloheximide for indicated timespan and the protein level of SETDB1 was detected by western blot. E The signal of (D) were quantified. F Cells from (A) were transfected with HA-Ub, treated with DMSO or MG132 for 4 h, then the ubiquitin level of SETDB1 was detected by western blot. G TRIM28-deficient U2OS cell lines were overexpressed with Vector control or SETDB1 and cell lysates were detected by western blot with the indicated antibodies. H Genomic DNA from cell lines (G) were used for the C-circle assay