Figure 6.
USP10 Knockdown Augments Cell Death Induced by a Proteasome Inhibitor
(A) USP10-KD (USP10-1) and control (NT) HeLa cells were treated with 5 μM MG-132 or DMSO for 12 hr, and the cells were stained with anti-cleaved caspase-3 (green) and anti-p62 (red) antibodies, and with Hoechst 33258 (blue). Scale bars, 10 μm.
(B) Apoptotic cells were assessed by staining cleaved caspase-3. Proportions of cleaved caspase-3-positive cells in USP10-KD (USP10-1) and control (NT) HeLa cells are presented as the mean ± SD (n = 3); **p < 0.01.
(C) USP10-KD (USP10-1) HeLa cells were treated with MG-132 or 1 μM bortezomib (BTZ) for 12 hr, and the cells were stained with the anti-p62 antibody and Hoechst 33258. Proportions of cells containing condensed nuclei with or without p62 aggregates are presented as the mean ± SD (n = 3); **p < 0.01; ***p < 0.001.
(D) HeLa cells were transfected with p62 small interfering RNA (siRNA) (p62-1 or p62-2) or control siRNA (NT) and cultured for 48 hr. Whole-cell extracts were characterized using western blot with anti-p62, anti-USP10, and anti-β-actin antibodies.
(E) USP10-KD (USP10-1) and control (NT) HeLa cells were transfected with p62 siRNA (p62-1 or p62-2) or control siRNA (NT), and further treated with MG-132 for 12 hr. Cells were stained with Hoechst 33258 (blue). The arrows indicate cells containing condensed nuclei (apoptotic cells). Scale bars, 10 μm.
(F) Proportions of cells containing condensed nuclei (apoptotic cells) are presented as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001.
(G) p62 fluorescence at aggresome (more than 15 μm2 in size) (p62-F at aggresome; n = 40) or the proportions of condensed nuclei (condensed nuclei [%]; n = 3) in USP10-KD (USP10-1) HeLa cells expressing wild-type USP10, USP10C424A, or USP1096−798 from Figures S5A or S5B are presented as the mean ± SD; *p < 0.05; ***p < 0.001; ****p < 0.0001.
See also Figure S5.