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. 2013 Nov 1;288(51):36437–36450. doi: 10.1074/jbc.M113.521344

FIGURE 2.

FIGURE 2.

NoxA1ds is cell-permeant and effective in whole colon adenocarcinoma cells yet ineffective in Nox1−/y cells. FITC-labeled NoxA1ds and native NoxA1ds were incubated with HT-29 colon adenocarcinoma cells. HT-29 cells bear the distinction of having abundant Nox1 expression while not expressing Nox2, Nox4, or Nox5. A–D, FITC-labeled NoxA1ds was incubated with HT-29 colon adenocarcinoma cells for 1 h prior to imaging. Confocal microscopy of these cells indicates that FITC-NoxA1ds penetrated the extracellular membrane and that its distribution is cytosolic and perinuclear. DIC, differential interference contrast. E, as measured by SOD inhibitable reduction of cytochrome c, increasing concentrations of NoxA1ds caused concentration-dependent inhibition of O2⨪ production by HT-29 cells. SCRMB peptide did not inhibit O2⨪ production, ***, p < 0.05 for 0.5, 1, and 5 μm NoxA1ds versus 0 μm NoxA1ds by one-way ANOVA. F, as measured by EPR, 10 μm NoxA1ds and Nox1 siRNA both significantly inhibited O2⨪ production by HT29 cells, and p values were determined by one-way ANOVA. G, Western blot analysis of Nox1/β-actin protein from Nox1 siRNA or control (Ctrl)-treated HT29 cells. H, peritoneal macrophages were isolated from Nox1 null mice, and O2⨪ production was measured by L-012 chemiluminescence after 2 h of incubation with NoxA1ds and subsequent PMA stimulation. No difference was observed between NoxA1ds-treated and -untreated samples. *, p < 0.05 for control versus siRNA, t test; AU is arbitrary units. All values are expressed as n = 9, except for H, which was quantified via three separate experiments; n = 10–12 cells from four individual mice.