Figure 5.

Effect of mARD1A²²⁵ on VEGFA expression. A) B16F10, B16F10-mock, and B16F10-mARD1A²²⁵ cells were exposed to 1% O₂ (4H) or 21% O₂ (N) for 4 hours (top). MKN74-mock and MKN74-mARD1A²²⁵ cells were exposed to 1% O₂ (4H) or 21% O₂ (N) for 4 hours (bottom). HIF-1α and mARD1A²²⁵ protein levels were examined by Western blot analysis. Anti-Myc antibody was used for the detection of mARD1A²²⁵. B) B16F10, B16F10-mock, and B16F10-mARD1A²²⁵ cells were exposed to 21% O₂ (N) or 10 μM MG132 and 1% O₂ for 6 hours (6H + MG132) (top). MKN74, MKN74-mock, and MKN74-mARD1A²²⁵ cells were exposed to 21% O₂ (N) (not shown) or 10 μM MG132 and 1% O₂ for 4 hours (4H + MG132) (bottom). Cell lysates were immunoprecipitated with an anti–acetyl lysine antibody and subjected to Western blot analysis with an anti-HIF-1α antibody. The level of mARD1A²²⁵ was determined using an anti-Myc antibody. C) MKN74, MKN74-mock, and MKN74-mARD1A²²⁵ cells were exposed to 1% O₂ (48H) or 21% O₂ (N) for 48 hours. Reverse transcription–polymerase chain reaction analysis was performed to detect gene expression using specific primers for VEGFA and glyceraldehyde 3-phosphate dehydrogenase. D) VEGFA protein levels were quantified in conditioned media from control and mARD1A²²⁵-expressing cells treated with CoCl₂ using enzyme-linked immunosorbent assay ELISA. B16F10, B16F10-mock, and B16F10-mARD1A²²⁵ cells were cultured in the absence (N) or presence of 200 μM CoCl₂ for 4 hours (4H) (left). MKN74, MKN74-mock, and MKN74-mARD1A²²⁵ cells were cultured in the absence (N) or presence of 200 μM CoCl₂ for 48 hours (48H) (right). Three independent experiments were performed, each in two replicates. Results are means and 95% confidence intervals (error bars) derived from the means of individual experiments (n = 3). *P = .046, **P = .05, ***P = .021, compared with mock control cells, determined with the two-sided Mann–Whitney U test.