FIGURE 6.

HDAC6 co-expression improves aggresome formation and rescue cell death in IBMPFD mutant-expressing cells. A, quantification of polyQ80-CFP-positive inclusion bodies (cells containing <3or ≥3 inclusions) in p97/VCP-WT or IBMPFD mutant-expressing (R155H, R95G, and A232E) cells with or without co-expression of HDAC6-FLAG. Note that all IBMPFD mutant cell lines have fewer cells with <3 inclusion than p97/VCP-WT. In addition, HDAC6 increases the number of cells with <3 inclusions in IBMPFD mutant-expressing cells. Non-HDAC6 transfected controls (<3 inclusions/cell) of each group versus HDAC6 transfected (<3 inclusions/cell) of each group have a p < 0.05 (^*). B, control U20S cells and U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP-R155H, -R95G, and -A232E were transfected with polyQ80-CFP with or without co-expression of HDAC6-FLAG. Forty-eight hours after transfection, the cell number was measured via MTT assay. Note that the number of IBMPFD mutant p97/VCP expressing is decreased compared with p97-VCP-WT, and HDAC6 increases the number of cells when co-expressed with IBMPFD mutant p97/VCP. Cell death is given as a ratio of the nontransfected controls of each group. The results are means ± S.E. and representative of at least three independent experiments. ^*, p < 0.05 versus non-HDAC6 overexpressing controls of each group. C, representative confocal micrographs of cells expressing p97/VCP-Myc, polyQ80-CFP, and HDAC6-FLAG. Note the co-localization of HDAC6 with a perinuclear inclusion body in IBMPFD mutant-expressing cells.