Fig. 2.

Carbonic anhydrase (CA) IX protein expression is decreased by 2-deoxy-d-glucose (2DG), pH 6.4, and SLC-0111 but not by hypoxia, while Na⁺/H⁺ exchanger-1 (NHE-1) expression remains constant. Wild-type and CA IX knockout (K/O) pulmonary microvascular endothelial cells (PMVECs) were seeded at 5.0 × 10⁵ cells/well on 6-well plates on bicarbonate-buffered media. Two days after cell seeding, media were changed to HEPES-buffered media with pH 7.4, 6.8, 6.6, and 6.4 and treated with 2DG (5 mM), DMSO (0.5%), and SLC-0111 (150 µM) in normoxia and hypoxia (1% oxygen). Twenty-four hours later, whole cell lysates were collected, and Western blotting was performed to assess CA IX (A and B) and NHE-1 (C and D) protein abundance. CA IX was decreased by 2DG, pH 6.4, and SLC-0111 but was not changed by hypoxia. NHE-1 expression remained unchanged across groups. Data represent means ± SD. Two-way ANOVA and Bonferroni post hoc tests were used to compare between different groups. At least five separate experiments were performed. *Significant difference (P < 0.05) from wild-type pH 7.4.