Fig. 1

TME conditions upregulate HIF-1α and VEGF – this is NHE1-independent whereas endothelial cell migration is dependent on NHE1. HUVECs or Ea.hy926 were grown under normoxic control (Ctrl), simulated tumor microenvironment (TME; 1% O₂, 1% FBS, 2.5 mM glucose, 7.5 mM lactate and pH 6.5) or hypoxic (Hyp; 1% O₂) conditions for 24 h, prior to cell lysis and western blotting with primary antibodies against HIF-1α, or RNA purification, reverse transcription and qPCR with primers against VEGFA₁₆₅. NHE1 was inhibited by cariporide (10 μM) or knocked down by siRNA treatment as indicated. a Representative western blot and quantification of HIF-1α protein levels after 24 h relative to the untreated control. GAPDH is shown as loading control. Quantified data are presented as means with SEM error bars of n = 3–5. ** indicate p < 0.01 compared to control cells, two-way ANOVA with Bonferroni’s multiple comparison post-test. The two-way ANOVA test also revealed a significant difference between conditions (Ctrl, TME, Hyp) with p < 0.0001. b, c Quantification of VEGF mRNA levels relative to the untreated control for HUVEC (B) and Ea.hy926 (C) cells. qPCR analysis was carried out as described in Methods, using GAPDH as housekeeping gene, and analysis was performed using the Pfaffl method. Data are shown as means with SEM error bars of n = 5. * denotes p < 0.05, one-way ANOVA with Tukey’s multiple comparison post-test. d Ea.hy926 cells were treated with NHE1 siRNA or scrambled control siRNA for 48 h (for knockdown efficacy, see Fig. 2d), whereafter a scratch in the culture was made with a sterile pipette tip and cell migration into the wound area monitored. Data are presented as means with SEM error bars of n = 3. The figure shows representative images and quantification of the wound area 18 h after scratch induction, relative to that of scrambled control siRNA