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Fig. 5 | BMC Cancer

Fig. 5

From: Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone: implications for endothelial cell function in cancer

Fig. 5

TME conditions regulate the phosphorylation levels of p70S6K, rpS6 and eIF2α. HUVECs or Ea.hy926 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, followed by lysis and western blotting. NHE1 was inhibited by cariporide (10 μM) or knocked down by siRNA-treatment where indicated. a Representative western blots of p-Thr389p70S6K and total p70S6K and quantification of p-p70S6K protein levels normalized to total p70S6K and relative to the untreated control condition for 24 h for HUVEC. GAPDH is shown as a loading control. b, c Representative western blots of p-Ser235/236rpS6 and quantifications of p-rpS6 protein levels relative to the untreated control condition for 24 h for HUVEC (b) and Ea.hy926 (c). GAPDH and p150 are shown as loading controls. d, e Representative western blots of p-Ser51eIF2α and quantifications of p-eIF2α protein levels relative to the untreated control condition for 24 h for HUVEC (d) and Ea.hy926 (e). p150 is shown as loading control. Data are presented as means with SEM error bars, with n = 5 except Hyp without/with cariporide for which n = 3. *, ** and *** indicates p < 0.05, p < 0.01 and p < 0.001, respectively, as obtained by two-way ANOVA with Bonferroni’s multiple comparison post-test. The two-way ANOVA test revealed a significant difference in p-p70S6K/p70S6K (p < 0.0001), p-rpS6 (p < 0.0001 for HUVEC and p < 0.05 for Ea.hy926) and p-eIF2α (p < 0.05 for HUVEC and p < 0.01 for Ea.hy926) between conditions (Ctrl, TME, Hyp). Also, p-p70S6K/p70S6K significantly changed with cariporide (p < 0.01)

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