Figure 6.

In vitro synergism of TSA/C6-ceramide in α-tubulin acetylation in cancer cell lines. CaOV3 or L3.6 cells were either left untreated or treated with indicated treatment (100 ng/ml of TSA, 10 μg/ml of C6-ceramide and TSA plus C6-ceramide) for 4 h, α-tubulin acetylation were detected by western blot assay by using commercially available antibodies (a and b). CaOV3 or L3.6 cells were either left untreated or treated as indicated (100 ng/ml of TSA, 10 μg/ml of C6-ceramide and TSA plus C6-ceramide) for 2 h, the association between HDAC6/α-tubulin were detected by immunoprecipitation assay as described (c–f). PP1 knockdown, HDAC6 knockdown, PP1/HDAC6 both knockdown L3.6 cells were treated with TSA (100 ng/ml) plus C6-ceramide (10 μg/ml) for indicated time points and ace-α-tubulin and α-tubulin were detected by western blot (g–i). The effects of PP1/HDAC siRNA knockdown (either alone or combined) on TSA (100 ng/ml) plus C6-ceramide (10 μg/ml) induced-L3.6 cell death/apoptosis were detected 48 h after treatment using MTT (j) and Histone-DNA ELISA assays (k) as described above. The values in the figures are expressed as the means±S.D. All experiments were repeated at least three times and similar results were obtained. ^*P<0.05 and ^#P<0.05 versus that indicated in the figure (ANOVA)