Fig 1: USP11 is Required for Chromatin Condensation and Genomic Stability. (A) U2OS cells were transfected with control or USP11 siRNAs, exposed to X-ray IR (10 Gy) and collected at different time points post IR. Nuclei were incubated with 2.5 U/ml MNase and purified and separated by electrophoresis in 1.2% agarose gel. Mono-, di-, tri- and upper nucleosome are indicated. (B) U2OS cells transfected with USP11 3′UTR siRNAs together with USP11 or USP11/C318S expression plasmids were exposed to X-ray IR (10 Gy) and subjected to MNase sensitivity assays at 8 h after irradiation. (C) U2OS cells treated with Mi-2β siRNAs and/or USP11 siRNAs were exposed to X-ray IR (10 Gy) and collected at 8 h post IR for MNase sensitivity assays. (D) U2OS clones with control or USP11 stably depleted were treated with DMSO or 2 μM CPT for 12 h, incubated in a final concentration of 1 μg/ml colcemid for 4 h followed by metaphase spreads preparation. Arrows indicate chromosome aberrations. (E) The percentage of chromosomal aberrations was quantified. Each bar represents the mean ± SD for triplicate experiments and at least 50 metaphases were counted in each experiment (*P < 0.05). (F) HeLa cells transfected with control or USP11 3′UTR siRNAs together with USP11 or USP11/C318S were treated with DMSO, CPT (1 μM, 48 h) or VP16 (40 nM, 48 h) and collected for annexin V and propidium iodide double staining. Cell apoptosis was determined by flow cytometry (right). Quantification was determined as mean ± SD for triplicate experiments (left, *P < 0.05). (G) HeLa clones with control or USP11 stably depleted were treated with X-ray IR at the indicated doses and subjected to clonogenic survival assays. Error bars represent mean ± SD for triplicate experiments (*P < 0.05). The representative images from biological triplicate experiments are shown. (H) U2OS clones with control or USP11 stably depleted were transfected with RNF20 or BMI1 siRNAs, and treated with IR at the indicated doses and subjected to clonogenic survival assays. Error bars represent mean ± SD for triplicate experiments (*P < 0.05). (I, J) U2OS clones with control or USP11 stably depleted were treated with CPT (I) or VP16 (J) at the indicated doses and subjected to clonogenic survival assays. Error bars represent mean ± SD for triplicate experiments (*P < 0.05). (K) U2OS cells were treated with control or USP11 siRNAs, and cell cycle profiles were analyzed by FACS.