Fig. 6

SOD3 stabilizes HIF-2α posttranscriptionally via NO. a Immunoblot analysis of 1G11-mock and -SOD3 cell extracts with anti-HIF-2α and -tubulin antibodies (n = 2). The ratio between bands is shown (bottom). b Immunofluorescence analysis of HIF-2α staining in mock and SOD3-expressing cells (n ≥ 500 cells/condition). c, d Percentage of nuclear area and mean fluorescence intensity in images in b. e Relative VEC promoter activity in IOX2- or Vhcl-treated 1G11-mock and -SOD3 cells. f Scheme of the ELISA used to detect PHD activity. g PHD activity, determined by ELISA, in mock and SOD3-expressing cell extracts. SOD3 levels are shown in these cells as determined by immunoblot (actin loading control; n = 3). h Top, HIF-2α ubiquitination analysis in immunoprecipitates of MG132-treated 1G11-mock and -SOD3 cells at the indicated times. Bottom, rehybridization with anti-HIF-2α antibody. i Immunoblot of HIF-2α in extracts from 1G11-mock and -SOD3 cells cultured in hypoxia (6 h), cycloheximide treated, and cultured in normoxia for the indicated times. Blots were rehybridized for tubulin as loading control. A representative experiment is shown (n = 4). j Quantification of the HIF-2α/tubulin densitometry ratio from i, expressed as the percentage of the ratio at time 0 (hypoxic conditions, 100%). k Representative images of Vhcl- or L-NMMA-treated 1G11-SOD3 cells stained with anti-HIF-2α antibodies. l, m Relative percentage of stained nuclear area and mean fluorescence intensity in Vhcl- or L-NMMA-treated 1G11-mock and -SOD3 cells. Values were normalized to the lowest value in the Vhcl-treated group for each cell type (n ≥ 100 cells/condition). For a, c–e, g, j, l, m, data shown as mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test (c, e, g, l, m) or two-way ANOVA with Bonferroni post-hoc test (j), showing significant differences between cell lines. Bar, 30 μm