AKT regulates glycolysis and OXPHOS in a FOXO-dependent fashion in MM cells. (A) Immunoblot analysis of HK2 and PKM2 expression in LME-1, MM1.S, and XG-3 Cas9-CTRL clones CTRL and FOXO KO clones (FOXO1 for LME-1, and FOXO3 for MM1.S and XG-3) treated overnight with 2.5 μM MK2206 or left untreated. β-actin served as a loading control. (B) Seahorse XF glycolysis stress test profiles of CTRL clones (left panels, n = 2), and FOXO knockout clones (middle panels, n = 2) treated for 20 hours with 2.5 μM MK2206 (red lines) or left untreated (blue lines). Means ± SEM of ECAR values are depicted (n = 5 measurements for each clone) and Seahorse XF injections are shown: glucose (=A), oligomycin (=B), 2-DG (=C). Bar graphs (right panels) depict basal glycolysis values, means ± SEM are shown (one-way ANOVA, with Bonferroni multiple comparison test). (C) Seahorse XF mitochondrial stress test profiles of CTRL clones (left panels, n = 2), and FOXO KO clones (middle panels, n = 2) treated for 20 hours with 2.5 μM MK2206 (blue lines) or left untreated (red lines). Means ± SEM of OCR values are depicted (n = 5 measurements for each clone) and Seahorse XF injections are shown: oligomycin (=A), FCCP (=B), rotenone and antimycin A (=C). Bar graphs (right panels) depict basal respiration values, and means ± SEM are shown (one-way ANOVA, with Bonferroni multiple comparison test, ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05; ns = not significant.