Fig. 3

TM blunts ribosomal protein S6 (S6) phosphorylation through the PI3K/Akt/mTOR pathway. a Treatment with increasing doses of TM at the indicated time-points prior to apoptosis induction decreased phosphorylation of Akt (p-Akt) at Ser 473, phospho-S6 (p-S6) at Ser235/6 and phospho-p70 S6 (p-p70 S6) in RPMI, B16F10, A375 and RPMI cells (representative immunoblots of n = 3–7 are shown) and quantified below. b Immunofluorescent p-S6 staining of B16F10 cells treated with TM for 2 h is shown (A representative image of n = 3 is shown) and quantified in B, lower panel). Scale bar indicates 50 μm. c Treatment for 48 h with the PI3K inhibitor ZSTK474 (2 μM for MeWo, 6 μM for MEL-JUSO and A375 and 1 μM for RPMI), pan-Akt inhibitor MK-2206 (2 μM for MeWo, 6 μM for MEL-JUSO, 10 μM for A375 and 4 μM for RPMI) and mTORC1/mTORC2 inhibitor KU-0063794 (2 μM for MeWo and 4 μM for all other cell lines) induced apoptosis in melanoma cells as assessed by Annexin V/7AAD staining (n = 3–6). Percent apoptosis was ascertained by summing up the Annexin V⁺/7AAD⁻ and Annexin V⁺/7AAD⁺. Error bars in the all experiments indicate SEM. *P < 0.05 as determined by a Student’s t-test (unpaired, 2 tailed) or a one-way ANOVA with a Dunnett’s post-hoc test