Fig. 2
NHE1 is downregulated by hypoxia and TME. HUVECs or Ea.hy926 were grown under 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 for 24 h (or 48 h as indicated in panel C). Subsequently, cells were lysed and subjected to SDS-PAGE and western blotting with primary antibodies against NHE1 or RNA purification, reverse transcription and qPCR with primers against NHE1 and GAPDH, as described in the Methods section. NHE1 was inhibited by cariporide (10 μM) as indicated. a NHE1 mRNA levels in HUVEC based on quantification of qPCR results relative to the untreated control and normalized to GAPDH levels. *** indicates p < 0.001, ANOVA with Tukey’s multiple comparison post-test. Data are shown as means with SEM error bars of n = 5. b Representative western blot and quantification (relative to Ctrl conditions), showing the protein expression levels of NHE1 in HUVEC after 24 h of TME or hypoxia exposure. GAPDH is shown as loading control. Quantified data are shown as means with SEM error bars of n = 3–5. c Representative western blot and quantification (relative to ctrl condition), showing the protein expression levels of NHE1 in HUVEC after 48 h of TME or hypoxia exposure. GAPDH is shown as loading control. Quantified data are shown as means with SEM error bars of n = 3. d, e Effects of NHE1 siRNA knockdown and TME conditions were evaluated using the Ea.hy926 cell line. Cells were treated with siRNA against NHE1 or scrambled control siRNA for 24 h prior to exposure to TME conditions. d NHE1 mRNA levels in Ea.hy926 quantified as in (A). Data are shown as means with SEM error bars, and n = 5. e Western blot analysis of NHE1 protein levels in Ea.hy926. p150 is shown as loading control. Representative of n = 3