FIG. 3.
Nox4 localizes to HUVEC ER. (A) TIRF imaging showing reticular distribution of Nox4-GFP on the ventral surfaces of HUVEC in comparison with the distribution of ssGFP-KDEL. Scale bars, 5 μm. (B) HUVEC cotransfected with Nox4-GFP (GFP fused to the Nox4 C terminus) and ssRFP-KDEL, imaged with TIRF optics. Scale bar, 20 μm. The expansions of the boxed area show green and red channels. (C) HUVEC cotransfected with GFP-Nox4 (GFP fused to the Nox4 N terminus) and ssRFP-KDEL, imaged with TIRF optics. Scale bar, 20 μm. (D) HUVEC lysate fractionated on a 5 to 30% discontinuous iodixanol gradient, immunoblotted for ER (calregulin and BiP), plasma membrane (Na-K ATPase and caveolin), or mitochondrial (HSP60) markers. Fractions 9 to 12 were enriched in ER markers. Fraction 5 contained a nonspecific low-density particulate fraction from multiple membrane sources. (E) HUVEC were infected with lentivirus containing control or Nox4 shRNA and then transfected with HyPer-ER and exposed to HeLa or HeLa-Tat cells for 3 days. Live-cell imaging was performed in the presence of cocultured cells. Representative fields are shown; scale bars, 20 μm. (F) HyPer ratios were compared in the preceding experimental groups. The means and SEM of 6 determinations are shown. *, P < 0.001 from control; †, P < 0.001 from HeLa-Tat with control shRNA. (G) HyPer ratios were obtained from HUVEC following lentiviral infection with the indicated shRNA and following exposure to vehicle (0.001% DMSO; veh) or tunicamycin (TM; 10 μg/ml) for 16 h. The means and SEM of 7 to 11 determinations are shown. *, P < 0.001 from control; †, P < 0.001 from TM with control shRNA. (H) HUVEC transfected with HyPer-ER were transduced with control adenovirus (lacZ) or Nox4 and p22phox for 3 days and/or treated with tunicamycin for 16 h, as indicated. The means and SEM of 17 to 25 determinations are shown. *, P < 0.01 from Ad-lacZ control; †, P < 0.01 from TM and Nox4/p22. TM alone was not different from Nox4/p22 (P > 0.05).