Fig. 3
TME conditions dramatically lower mRNA and protein levels of Akt1 in HUVEC and Ea.hy926 cells, associated with increased relative phosphorylation of Akt. Cells were exposed to normoxic control (Ctrl), TME (1% O2, 1% FBS, 2.5 mM glucose, 7.5 mM lactate and pH 6.5) or hypoxic (Hyp; 1% O2) conditions as indicated, for 24 h before lysis and RNA purification, reverse transcription and qPCR or western blotting, as indicated. a Relative mRNA levels of the three Akt isoforms Akt1–3 in HUVEC (left panel) and Ea.hy926 (right panel) under Ctrl conditions. b Relative mRNA levels of Akt1–3 in HUVECs exposed to Ctrl, TME or Hyp conditions. Data are shown as means with SEM error bars and n = 5. * and ** denotes p < 0.05 and p < 0.01, respectively, one-way ANOVA with Tukey’s multiple comparison post-test. c Akt1 and p-Ser473Akt levels in HUVEC cells after 24 h of Ctrl, TME or Hyp conditions in the absence or presence of 10 μM cariporide. Top: representative western blots (GAPDH is shown as loading control), middle: protein level of total Akt1, bottom: p-Ser473Akt normalized to total Akt1. d As C, except for Ea.hy926 cells treated with NHE1 siRNA or scrambled control siRNA, and exposed to Ctrl or TME conditions. p150 is shown as loading control. Data are shown as means with SEM error bars, relative to control, and n = 3 for Hyp conditions, n = 5 for all other conditions. *** denotes p < 0.001, two-way ANOVA with Bonferroni’s multiple comparison post-test. The test revealed a significant difference in Akt1 protein levels between conditions (Ctrl, TME, Hyp), p < 0.01 for HUVEC and between conditions (Ctrl, TME), p < 0.0001 for Ea.hy926 cells