Figure 2

PI3K-AKT inhibitors enhancement of vorinostat (vo)-induced cytotoxicity is mediated by strong and durable AKT inhibition. (A) LDH viability assay for SCC25 cells treated with vehicle (CTR) or a maximal cytocidal dose of vo (5 μ M) alone or in combination with LY (10 μ M) or uo (10 μ M) for 24 h. (B, C) Western blots show lysates from SCC25 cells treated with vo (5 μ M) alone or in combination with LY (10 μ M) or uo (10 μ M) at distinct time points. (D) LDH viability assay for Cal27 cells treated with vehicle (CTR) or vo (5 μ M) alone or in combination with LY (10 μ M) or uo (10 μ M) for 24 h. (E) LDH viability assay for SCC25 cells treated with valproic acid (VA, 3 mM) or Depsipeptide (Depsip, 5 nM) alone or in combination with LY (10 μ M). (F) SCC25 cells were transfected with constitutively active AKT (myr-AKT, black boxes) or its corresponding empty vector (open boxes) and then left untreated (CTR) or were treated for 24 h with vo (5 μ M)+LY294002 (10 μ M, LY). Cell viability was then estimated by LDH release. (G) Western blot of lysates from SCC25 cells showing the expression of the myr-AKT and endogenous AKT in vector only and myr-AKT transfected cells. Phosphorylation of the AKT target GSK3β (p-GSK3β) are provided to confirm functional AKT activity. Western blot figures are representative of two independent experiments. (H) LDH viability assay for SCC25 cells treated with vo (5 μ M) alone or in combination with LY (10 μ M) or wortmannin (1 μ M). (I) Similar assay for cells treated with vo (5 μ M) alone or in combination with AKTVIII (10 μ M) for 24 h. LDH values presented as a percent of total LDH. Western blot figures are representative of at least three independent experiments. ^*Indicates P⩽0.05. ^**Indicates P⩽0.01. ^***Indicates P⩽0.001 vs CTR. Values presented as mean±s.e.m. of at least three independent experiments performed in triplicate.