Figure 1.
USP10 Knockdown Impairs Aggresome Formation
(A) HeLa cells were treated with 5 μM MG-132 or DMSO for 12 hr, and the cells were stained with anti-HDAC6 (green) or anti-USP10 (green) antibody with either the anti-p62 (red) or anti-ubiquitin (Ub) (red) antibody. Nuclei were counterstained using Hoechst 33258 (blue). Arrows indicate cells with USP10/p62-double-positive aggregates. Scale bars, 10 μm.
(B) HeLa cells were pretreated with 2.5, 5, and 10 nM bafilomycin A1 (BafA1) or DMSO for 0.5 hr and further treated with MG-132 or DMSO for 12 hr. The whole-cell extracts were characterized by western blot (WB) analysis using anti-USP10, anti-LC3, and anti-β-actin antibodies.
(C) USP10-KD (USP10-1 or USP10-3) and control (NT) HeLa cells were treated with MG-132 or DMSO for 12 hr, and the cells were stained with anti-HDAC6 (green) and anti-p62 (red) antibodies and with Hoechst 33258 (blue). Scale bars, 10 μm.
(D) The indicated HeLa cells were treated with MG-132, 1 μM bortezomib (BTZ), or DMSO for 12 hr. Cells with one large HDAC6/p62-double-positive aggregate (more than 15 μm2 in size) at the perinuclear region with nuclear deformity were counted as aggresome-positive cells. Cells with multiple small HDAC6-negative/p62-positive aggregates (less than 10 μm2 in size) were counted as p62-aggregate-positive cells. The number of cells with p62 aggregates or aggresome are presented as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
(E) Whole-cell extracts prepared from the indicated HeLa cells were characterized by WB using anti-USP10, anti-G3BP1, anti-Ub, anti-p62/pS349, anti-p62, anti-LC3, and anti-β-actin antibodies.
(F) The indicated HeLa cells were pretreated with increasing concentrations of BafA1 (–, DMSO; +, 5 nM BafA1; ++, 10 nM BafA1) and further treated with MG-132 or DMSO for 12 hr. The whole-cell extracts were characterized by WB using anti-LC3, anti-p62/pS349, anti-p62, anti-Ub, and anti-β-actin antibodies.
See also Figure S1.