Figure 5

In vitro synergism of TSA/C6-ceramide in AKT/mTOR inhibition. 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 12 h; AKT and downstream mTOR activation 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 with indicated treatment (100 ng/ml of TSA, 10 μg/ml of C6-ceramide and TSA plus C6-ceramide) for 2 h, the association between PP1/HDAC6/AKT1 were detected by immunoprecipitation (IP) assay as described (c and d). CaOV3 or L3.6 cells were pretreated with PP1-specific inhibitor PP1 i2 (5 μM) or PP2A-specific inhibitor Cantharidin (5 μM) for 1 h, followed by TSA/C6-ceramide treatment for another 12 h, p-AKT (Ser 473) and AKT were detected by western blot (e and f). CaOV3 or L3.6 cells were either left untreated or treated with indicated treatment (100 ng/ml of TSA, 10 μg/ml of C6-ceramdie and TSA plus C6-ceramide) for 2 h, PP1 activity was detected as described (g and h). L3.6 cells were transfected with control siRNA (scramble, 250 nM), PP1 siRNA (250 nM), HDAC6 siRNA (250 nM), PP1+HDAC6 (250 nM each) for 72 h (to reach maximum transfection efficiency) using RNAi methods as aforementioned, expression level of PP1 and HDAC6 were measured using western blot. Successfully knockdown cells were used for further experiment. PP1/HDAC single (i and j) or double (k) knockdown L3.6 cells were treated with TSA (100 ng/ml) plus C6-ceramide (10 μg/ml) for indicated time points; p-AKT, actin, PP1 and HDAC6 were detected by western blot. (l) L3.6 cells transfected with empty vector or Myc-D95N-PP1 plasmids (1 μg/well, described in materials and methods) were treated with TSA (100 ng/ml) plus C6-ceramide (10 μg/ml) for 24 h and AKT/mTOR activation was detected using specific antibodies. Data are presented as the mean±S.D. for three independent experiments. ^*P<0.05 versus that indicated in the figure (ANOVA). All experiments were repeated at least three times and similar results were obtained