Localization of MDR-1 (P-gp) protein in HIV-1 and SQV coexposed HBMVEC.

<p>A representative immunofluorescence microscopy data of MDR-1 protein in HBMVEC following exposure to HIV-1 or SQV for 24 hrs is shown. Green punctate MDR-1 staining appeared in (<b>A</b>) untreated cells, (<b>B</b>) cells exposed to SQV (1 µM), (<b>C</b>) cells exposed to HIV-1 (0.2 MOI) only, and (<b>D</b>) combined exposure of SQV and HIV-1. DAPI (blue) staining of the nucleus and WGA (red) staining of cell plasma membrane are also shown. Image Magnification: 630X, scale bar: 21 µm.</p

Effect of HIV-1 exposure on MDR1 gene expression and efflux function in HBMVEC.

<p>(<b>A</b>) MDR-1, MRP-1 and MRP-2 gene expression following exposure to HIV-1 (0.2 and 1.0 MOI) within 6 hrs post exposure was monitored by qRT-PCR. Data obtained in each sample was normalized to GAPDH mRNA levels (internal control). Fold change in ABC-transporter gene expression with respect to that observed with MDR1 in untreated cells (1.0) was calculated under each treatment condition and for each transporter. Data represent three independent experiments (n = 3) where standard deviation (error bars) were calculated from triplicate samples and significant differences from controls are shown (*, p<0.05 and ** p<0.01). (<b>B</b>) MDR1 efflux function following exposure to HIV-1 (0.2 and 1.0 MOI) for 24 hrs. Verapamil and MK-571 were used as MDR1 and MRPs blocker, respectively. Intracellular fluorescence was normalized to total protein content in cell lysates and change in calcein-AM retention is shown in the bar graphs (*, p<0.05). (<b>C</b>) Comparative effects of conditioned medium (CM) from HIV-1-infected or PMA-stimulated HuT-78 T-cells on MDR1 gene expression. The RT-PCR products from HBMVEC control (1); Hut-78 CM (2); CM from HuT-78 cells treated with PMA (3); or CM from virus producing HTLV-IIIB cells (4), are shown. (<b>D</b>) Effect of live HIV-1 (HTLV-IIIB CM) and heat inactivated HIV-1 (HTLV-IIIB CM-HI) on MDR1 gene expression. The respective fold change in MDR1 gene expression was calculated with respect to GAPDH (internal control). A representative data from three independent experiments, is shown.</p

Effect of MDR1 specific siRNA on HIV-1 and SQV induced drug efflux in HBMVEC.

<p>(<b>A</b>) Fold change in ³H-SQV retention (CPM/µg of protein) in HBMVEC are shown in control (untreated) and HIV-1 (0.2 MOI) and SQV (0.3 µM) exposed cells. Data from non-transfected, and in cells transfected with either a random (NS) or an MDR1 specific siRNA, are shown. Data are representative of three independent experiments carried out in triplicates (*, p<0.05 and **, p<0.001). (<b>B</b>) Relative inhibition of MDR1 gene expression in MDR1 siRNA transfected and control (random) siRNA transfected cells are shown by semi-quantitative RT-PCR. Change in MDR1 gene expression following treatment with HIV-1 (0.2 MOI) and HIV-1+SQV, in control and siRNA transfected cells, are shown. Changes in band intensities of PCR products for MDR1 mRNA were normalized to the GAPDH mRNA levels, in respective samples, and fold changes obtained compared to control (1.0) are shown.</p

A comparative analysis of ³H-SQV efflux in HBMVECs exposed to HIV-1 and/or SQV in presence of verapamil or PSC-833.

<p>(<b>A</b>) Effect of verapamil (50 µM) on ³H-SQV retention in HBMVEC exposed to HIV-1 and/or SQV for 24 hrs. Under each treatment condition, intracellular ³H-SQV was measured in presence of verapamil and compared with control (without inhibitor). The radioactivity in cell lysates were normalized to total cell protein content (CPM/ug of protein) and data from three independent experiments are presented as fold changes. (<b>B</b>) Effect of increasing concentrations (5–50 µM) of verapamil or PSC-833 on ³H-SQV retention in HBMVEC exposed to HIV-1 and SQV for 24 hrs. Data are representative of three separate independent experiments carried out in triplicates (*, p<0.05 and **, p<0.01).</p

Effect of HIV-1 and SQV coexposure on MDR1 expression and function in HBMVEC.

<p>(<b>A</b>) MDR1 gene expression in presence of HIV-1 (0.2 MOI) and increasing concentration of SQV (0.3 and 1.0 µM). (<b>B</b>) Densitometric analysis of mRNA expression from Fig. 2A. (<b>C</b>) A representative of western blot analysis data of MDR1 protein expression in untreated (1), HIV-1 (2), SQV (3) and HIV-1+ SQV (4) exposed cells. Beta (β)-actin was used as internal control. The fold change analysis was done from three sets of independent western immunodetection studies. MDR1 protein expressions are normalized with β-actin expression in respective samples. (<b>D</b>) MDR1 mediated calcein-AM efflux in presence of HIV-1 (0.2 MOI) and/or SQV (0.3 µM). Grey bars indicate calcein retenition without inhibitor and black bars indicate calcein retention in presence of 50 µM verapamil. Data shown are mean of three independent experiments (*, p<0.05 and **, p<0.01).</p