Fig. 4
From: SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2α
SOD3-induced VEC expression requires SOD3 enzyme activity and NO. a VEC and tubulin (loading control) levels in 1G11-mock- and -SOD3-transduced cells. The VEC/tubulin ratio is indicated (n = 3). b VEC mRNA normalized to CD31 levels in mock and SOD3-expressing cells (n = 5). c VEC staining (red) in 1G11-mock- and 1G11-SOD3-transduced cells. Nuclear staining with DAPI (blue) (n > 20 fields/condition). d Continuous vs. discontinuous junctions expressed as junction length (% total junction length) in mock and SOD3-expressing cells. e Gap index in confluent cells as in c. (d, e, n ≥ 5 fields/condition). f FITC-dextran (40 kDa) permeability of 1G11-mock, -SOD3 and -SOD3S195C cell monolayers (n = 3). g SOD3 levels in cells as in f; bottom, filter rehybridized with actin. SOD3/actin ratio indicated (n = 3). h FITC-dextran permeability of 1G11-mock and -SOD3 monolayers, untreated or VEGF-pretreated (n = 9). i VEC staining of untreated and VEGF-treated cells as in h. j, k Continuous vs. discontinuous junctions (j) and gap index (k) in VEGF-treated and untreated cells; n ≥ 5 fields/condition. l FACS analysis of DAF2 staining in 1G11-mock and -SOD3 cells. Non-specific staining (gray). m DAF2 intensity from cells as in l (n = 3). n NO distribution in LLC tumors from Lov-treated WT and SOD3−/− mice, visualized by DAR-1, microangiography using FITC-lectin, merge of DAR-1, FITC-lectin, and DAPI counterstaining, and pseudocolor representation of DAR-1 microfluorographs. Arrowheads indicate large blood vessels. Images are confocal microscopy z-stacks. o eNOS detection in sections from tumors as in n. p FITC-dextran permeability of L-NMMA- or Vhcl-pretreated 1G11-mock and -SOD3 monolayers (n = 6). q VEC promoter activity in L-NMMA- or Vhcl-treated 1G11 cells (n = 5). r VEC promoter activity in DETA-NONOate- or Vhcl-treated 1G11 cells (n = 9). s FITC-dextran permeability of DETA-NONOate-pretreated 1G11-mock and -SOD3 monolayers; 0 = Vhcl (n = 9). Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test (b, d, e, k, m, p–r) or ANOVA with Dunnett’s (f, s) or with Tukey’s (h, j) post-hoc tests. Bar, 5 μm (c) and 25 μm (i, n)
