Figure 2.
Overexpression of USP10 Stimulates Aggresome Formation
(A) HeLa cells were transfected with the GFP-CFTR-ΔF508 plasmid and treated with 5 μM MG-132 or DMSO for 12 hr. The cells were stained with the anti-HDAC6 antibody (red) and Hoechst 33258 (blue). Arrows indicate localizations of HDAC6 at CFTR-ΔF508-induced aggresomes. Scale bars, 10 μm.
(B) HeLa cells were transfected with the HA-tagged USP10 (HA-USP10) and GFP-CFTR-ΔF508 plasmids, and the cells were treated with MG-132, 10 nM BafA1, or DMSO for 6 hr. Cells with GFP/HDAC6-double-positive aggresomes (more than 15 μm2 in size) at the perinuclear region with nuclear deformity were counted as aggresome-positive cells. The percentages of cells with GFP-positive aggresome are presented as the mean ± SD (n = 3); ****p < 0.0001; NS, not significant. Simultaneously, whole-cell extracts were characterized by western blot (WB) using anti-GFP, anti-HA, and anti-β-actin antibodies.
(C) USP10-KD (USP10-1) and control (NT) HeLa cells were transfected with the HA-USP10 and the GFP-CFTR-ΔF508 plasmids. The percentages of cells with GFP/HDAC6-positive aggresome are presented as the mean ± SD (n = 3); *p < 0.05; **p < 0.01. Simultaneously, whole-cell extracts were characterized by WB using anti-GFP, anti-HA, anti-USP10, and anti-β-actin antibodies.
(D) HeLa cells were transfected with the HA-USP10 plasmid with or without the GFP-CFTR-ΔF508 plasmid, and the whole-cell extracts (WCE), NP-40-soluble fractions (SF), and NP-40-insoluble fractions (ISF) were characterized by WB using anti-GFP, anti-Ub, anti-HA, anti-Lamin B, and anti-β-actin antibodies.
(E) USP10-KD (USP10-1) HeLa cells were transfected with HA-USP10 or its deubiquitinase-inactive mutant USP10C424A plasmid together with the GFP-CFTR-ΔF508 plasmid. The percentages of cells with GFP/HDAC6-positive aggresome are presented as mean ± SD (n = 4); **p < 0.01; ***p < 0.001; NS, not significant. Simultaneously, whole-cell extracts were characterized by WB using anti-GFP, anti-HA, and anti-β-actin antibodies.
(F) USP10-KD (USP10-1) HeLa cells were transfected with HA-USP10 or its mutant (USP101−274, USP101−214, or USP10275−798) plasmid together with the GFP-CFTR-ΔF508 plasmid. The percentages of cells with GFP/HDAC6-positive aggresome are presented as the mean ± SD (n = 4); *p < 0.05; ***p < 0.001; ****p < 0.0001; NS, not significant. Simultaneously, whole-cell extracts were characterized by WB using anti-GFP, anti-HA, and anti-β-actin antibodies.
(G) Schematic representation of human USP10 mutants used in this study.
See also Figure S2.