S6K1i inhibits S6 phosphorylation and synergizes with MCL-1i in apoptosis induction of HMCLs, independent of BIM protein induction. (A) Western blot showing total S6 and S6 phosphorylation on serine residues 235/236 (pS6 S235/6) and 240/244 (pS6 S240/4) in MM1.s after 4 hours of exposure to indicated concentrations of S6K1i. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Histograms comparing expression of S6 phosphorylation on serine residues 235/236 (pS6 S235/236) and 240/244 (pS6 S240/244) in MM1.s after 4 hours of exposure to 1 μM dexamethasone (DEX) or 10 μM S6K1i, determined by flow cytometric analysis. Expression was relative to medium-treated control cells (ctr) and corrected for isotype control, indicated by the dashed line. (C) Heatmaps showing specific apoptosis of indicated HMCLs induced by serial dilution of S6K1i and MCL-1i, individual or combined. Viability was analyzed after 48 hours of drug exposure; values show the mean of 3 individual experiments. (D) Plots comparing expected (EXP) specific apoptosis vs observed (OBS) specific apoptosis induced by S6K1i and MCL-1i combinations. Per HMCL, the drug combination that resulted in the highest average OBS-EXP ratio was selected from the data obtained in panel C. The 3 connected data points show the data obtained from 3 individual experiments. Statistical analysis was performed by paired Student t tests. (E) Representative western blot showing expression of 3 BIM isoforms in indicated HMCLs after 24 hours of exposure to 1 μM DEX, 10 μM S6K1i, or negative control. α-tubulin was used as a loading control. (F) Comparison of BIM isoform expression in HMCLs treated with 1 μM DEX or 10 μM S6K1i. Values show the mean quantification of 3 individual western blot experiments, normalized to α-tubulin and relative to untreated ctr cells, indicated by the dashed line. Statistical analysis was performed by two-way analysis of variance using Šidák correction for multiple testing. ns, not significant.