Fig. 2: USP10 stabilizes HDAC6.

a 293T cells were transfected with empty vector, wild-type USP10 (Flag-USP10), catalytically-dead mutant of USP10 (Flag-USP10CA) or other DUBs as indicated. Thirty-six hours post-transfection, cells were harvested. Anti-HDAC6, anti-USP10, anti-DUBs, and anti-β-actin Western blotting analyses were performed. b Depletion of USP10 reduces the level of HDAC6. Six lung and ovarian cancer cell lines, H157, H125, H1299, H23, SKOV3, and ES-2, were infected with lentivirus encoding two shRNAs against USP10. The anti-USP10, anti-HDAC6, and anti-β-actin Western blotting analyses were performed as indicated. c Depletion of USP10 does not affect the mRNA level of HDAC6 in H23 cells. Total RNAs extracted from shcontrol, shUSP10-1 or shUSP10-2 lentivirus stably infected H23 cells were subjected to RT-PCR for HDAC6, USP10, and GAPDH, as indicated. d HDAC6 protein levels are positively correlated with USP10 in a panel of lung and ovarian cancer cell lines. HDAC6, USP10, and β-actin protein levels were obtained by Western blotting analyses of 17 cell lines (A549, A549-USP10KD, EPLC, H292, H1299, H1975, H522, H661, CAOV3, OVCAR3, TOV21G, SKOV3, CHI, CHI-CisR, M41, PEO1, and DOV). e HDAC6 and USP10 protein levels were normalized by the level of β-actin and quantified with Image Pro Plus 6.0 software. The correlation analysis was performed by Excel CORREL function and the results were shown as a scatter plot indicating a strong positive correlation coefficient of USP10 and HDAC6 (r = 0.78, p < 0.01). f The half-life of HDAC6 is shortened in USP10KO MEFs. Cycloheximide (CHX) was added to USP10 WT and USP10 KO MEFs at the indicated concentration and time intervals. The anti-HDAC6, anti-USP10, and anti-β-actin Western blotting analyses were performed (top panel). The experiments were repeated three times. The HDAC6 expression was quantified and a plot showing half-life of HDAC6 in USP10 WT and USP10 KO MEFs was drawn (lower panel). g The half-life of HDAC6 is shortened in USP10KD H23 cells. Half-lives of HDAC6 in H23-shcontrol and H23-shUSP10 cells were performed as f. h USP10 inhibitor Spautin-1 shortens the half-life of HDAC6. Spautin-1 was added to the H23 cells at the indicated concentrations and time intervals. The anti-HDAC6, anti-USP10, and anti-β-actin Western blotting analyses were performed. Half-life of HDAC6 in DMSO-treated or Spautin-1-treated H23 cells was performed as f. For graphs in f–h, the mean band intensities from three independent experiments as measured by Image-Pro plus 6.0 shows the approximate half-lives in the presence of CHX. The error bars represent the standard deviation.