Figure 2.

MG132-induced centrosome accumulation of parkin is decreased by HDAC6 siRNA and increased by HDAC6 overexpression. A–C, SH-SY5Y cells cotransfected with GFP and siRNA for HDAC6 or control siRNA with scrambled sequence were treated without or with MG132 (5 μm for 12 h). Cells were stained for parkin (PKN, green). GFP as a transfection marker was pseudocolored red. HDAC6 siRNA (H6-siRNA) markedly reduced centrosomal accumulation of parkin in response to MG132 (B), while the control siRNA (Con-siRNA) had no significant effect (A). Scale bar, 10 μm. Arrow, Parkin accumulation in transfected cells. C, Statistical summary of the intensity of parkin accumulation under the above conditions and in the absence of MG132. *p < 0.001 versus Con-siRNA + MG132, n = 30 cells randomly selected from three independent experiments for each condition. D, Transfection of HDAC6 siRNA (marked by GFP, green) greatly diminished HDAC6 expression as indicated in immunofluorescence (red) and immunoblot (inset). E–G, SH-SY5Y cells cotransfected with GFP and wild-type HDAC6 (wt-H6, E and G) or its catalytically inactive double mutant (mut-H6, F) were treated with MG132 (MG, 5 μm for 12 h) in the absence or presence of TBC (10 μm, G). Cells were stained for parkin (PKN, green) and GFP fluorescence (marking transfected cells) was pseudocolored red. As highlighted by arrows, MG132-induced parkin accumulation was markedly increased in cells transfected with wt-H6 (E), but not mut-H6 (F). The effect of wt-H6 was reversed by tubacin (G), but not niltubacin (H). H, Statistical summary of the intensity of parkin accumulation at the centrosome under the conditions in (E–G). *p < 0.002 versus empty vector (Vec) transfection plus MG132 treatment; ^#p < 0.001 versus wt-H6 plus MG132; n = 30 cells randomly selected from three independent experiments for each condition.