HIV-1 Tat C upregulates NOX2 and NOX4 expression and increases ROS production in hBMVECs. (A) Western blot analysis to monitor the changes in expression levels of NOX2 and NOX4 in hBMVECs exposed to HIV-1 Tat C protein. The level of expression of both the NOX proteins increased after HIV-1 Tat C treatment, and led to enhanced ROS production by hBMVECs in a dose–dependent manner. (B, C) Densitometry for western images for NOX2 and NOX4 normalized with image density of β-tubulin. The graphs are representative of three independent experiments and results are shown as mean ± S.E. The levels of significance in the changes of the gene expression; compared to control groups, the p values ≤0.05 are indicated as *above the bar, (*for p value ≤0.05, **for p value ≤0.005). (D) The figure showing fluorescence microscopic images of intracellular ROS level in hBMVECs detected via DCFDA staining. Increasing doses of HIV-1 Tat C treatments are showing increase in ROS level in linear fashion. hBMVECs were treated with HIV-1 Tat C protein, washed with HBSS and incubated with DCFDA reagent (10 μM final concentration) for 15 minutes, washed and visualized under microscope. Images were captured by Axio Observer-A1 at 20 × magnification with GFP filters.