Fig. 5.

2-Deoxy-d-glucose (2DG) decreases and extrinsic acidosis increases Matrigel network master junction number and total segment length, and 2DG modifies N-cadherin expression. A–D: 96-well plates were loaded with Matrigel, 30 µl per well, and incubated at 37°C with 0% CO₂ room air for 1 h. Wild-type and carbonic anhydrase (CA) IX knockout (K/O) cells were seeded at a density of 4.0 × 10⁴ cells per well in bicarbonate-free HEPES-buffered media with a range of pH and inhibitors and a total cell solution volume of 100 µl per well. Cells were incubated at 37°C with 0% CO₂ room air for 24 h. Pictures were taken at 24 h and analyzed using ImageJ. Total master junction number and segment length were decreased by 2DG and increased by extrinsic acidosis. E and F: wild type and CA IX 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 was 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 N-cadherin protein abundance. N-cadherin expression was increased, and its molecular weight was decreased by 2DG. 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.