Figure 8.

Effects of lysine 532 mutation in HIF-1α. A) HIF-1α protein levels were determined by Western blot analysis. B16F10-mock, B16F10-mARD1A²²⁵, and B16F10-mARD1A²²⁵-HIF-1α/K532R cells were cultured in the absence (N) or presence of 200 μM CoCl₂ for 24 hours (24H). Similar results were obtained with three independent experiments. B) VEGFA levels (in picograms per milliliter) were quantified using enzyme-linked immunosorbent assay. B16F10, B16F10-mock, B16F10-mARD1A²²⁵, and B16F10-mARD1A²²⁵-HIF-1α/K532R cells were cultured in the absence (N) or presence of 200 μM CoCl₂ for 4 hours (4H). Three independent experiments were performed, each in two replicates. Results are means and 95% confidence intervals (error bars) derived from the means of the individual experiments (n = 3). *P = .05 for all comparisons as determined with the two-sided Mann–Whitney U test. C) B16F10, B16F10-mock, B16F10-mARD1A²²⁵, and B16F10-mARD1A²²⁵-HIF-1α/K532R cells were grown to 80% confluence. A linear injury line was created at the center of the cultured monolayer by scraping with a sterile pipette tip. Cells were incubated in the presence of 200 μM CoCl₂ for 24 hours. B16F10, B16F10-mock, and B16F10-mARD1A²²⁵-HIF-1α/K532R cells were treated with 3 μg/mL anti-mouse VEGFA antibody. B16F10-mARD1A²²⁵ cells were treated with 25 ng/mL recombinant mouse VEGFA protein. Images of cell migration (left). Quantification of cell migration (right). Migration distance (in micrometers); P = .004, determined with the two-sided Mann–Whitney U test. Three independent experiments were performed, each in two replicates. Results are means and 95% confidence intervals (error bars) derived from the means of the individual experiments (n = 3). Magnification ×100. Scale bar = 100 μm. D) C57BL/6 mice were injected intravenously with B16F10-mock (n = 40 mice), B16F10-mARD1A²²⁵ (n = 31 mice) and B16F10-mARD1A²²⁵-HIF-1α/K532R cells (n = 11 mice). Lung nodule formation was determined 14 days after injection. Representative gross images of lungs are shown (left). Numbers of surface tumor nodules per mouse (right). Results are means and 95% confidence intervals (error bars). *P < .001, compared with the number of surface tumor nodules in B16F10-mock–injected mice, #P < .001, compared with the number of surface tumor nodules in B16F10-mARD1A²²⁵–injected mice, as determined with the two-sided Mann–Whitney U test. A#1 = B16F10-mARD1A²²⁵ #1; A#2 = B16F10-mARD1A²²⁵ #2; A#1/HIF K532R = B16F10-mARD1A²²⁵ #1-HIF-1α/K532R; B16F10 + anti-VEGFA = B16F10 + anti-mouse VEGFA antibody; Mock + anti-VEGFA = B16F10-Mock + anti-mouse VEGFA antibody; A#1/HIF K532R + anti-VEGFA = B16F10-mARD1A²²⁵ #1-HIF-1α/K532R + anti-mouse VEGFA antibody; A#1+VEGFA = B16F10-mARD1A²²⁵ #1 + recombinant mouse VEGFA protein; A#2+VEGFA = B16F10-mARD1A²²⁵ #2 + recombinant mouse VEGFA protein.