Figure 1. Hypoxia increases NHE-1 activity in HT-1080 cells.

(A) Control (untransfected) HT-1080 cells incubated in the presence or absence of the NHE-1 inhibitor, EIPA (25 µM), or cells transfected with scrambled (control) shRNA, or NHE-1 shRNA, were incubated for 4 h under normoxic or hypoxic conditions. Basal intracellular pH (pH_i) was measured using BCECF (n = 300 cells). Immunoblot showing knock down of NHE-1 expression in NHE-1 shRNA transfected cells. Actin was used as a loading control. (B–D) Representative traces showing pH_i recovery after ammonium chloride-prepulse-induced acidification in HT-1080 cells cultured under normoxic or hypoxic conditions with associated bar graph showing Na⁺/H⁺ exchange rates (n = 300 cells per condition), for (B) untransfected cells, (C) cells transfected with p-NHE-1, or (D) cells transfected with control shRNA or NHE-1 shRNA. (E) Rescue experiments were performed by transfection of NHE-1 shRNA-transfected cells with pNHE-1. Na⁺/H⁺ exchange rates were measured in cells exposed to normoxic or hypoxic conditions. Columns represent the mean ± SEM indicated by the horizontal bars. The asterisks correspond to, * p<0.01; ** p<0.001; *** p<0.0001.