Fig. 4.
Extrinsic acidosis and carbonic anhydrase (CA) IX inhibition suppresses migration. 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, monolayers were scratched with a sterile 200-μl pipette tip. Media were changed to HEPES-buffered media with pH 7.4, 6.8, 6.6, and 6.4 and treated with 2-deoxy-d-glucose (2DG; 5 mM), DMSO (0.5%), and SLC-0111 (150 µM) in normoxia and hypoxia (1% oxygen). Baseline and 24-h time point wounds were imaged and analyzed using ImageJ. At baseline, there was no difference in wound areas across groups (A and B). At 24 h, wound areas were bigger in extrinsic acidosis and CA IX inhibition (C and D). On separate experiments, cells were treated with DMSO (0.5%), pH 6.4, SLC-0111 (150 µM), and staurosporine (500 nM), lysates were collected after 24 h, and caspase-3 activity assays were performed. pH 6.4 or SLC-0111 had no effect on caspase-3 activity. 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; ^significant difference (P < 0.05) from CA IX K/O pH 7.4.