Knockdown of AR expression decreases the efficiency of KSHV infection in endothelial cells.

<p>(a) Endogenous AR expression in HUVECs and SLK cells is efficiently impaired by RNA interference of the AR. siRNAs were transfected into cells at final concentrations of 100 nM for HUVECs and 200 nM for SLK cells using Lipofectamine 2000. Cell lysates were subjected to immunoblotting using the indicated antibodies. (b, c). RNA transcription of the AR and its target genes is inhibited by AR siRNA treatment in both HUVECs (b) and SLK cells (c). Total RNA from the above samples was subjected to a qRT-PCR to determine the mRNA levels of the indicated genes. Values were normalized to the level of actin mRNA and calibrated by comparison to a control siRNA treatment. (d) AR inhibition leads to a significant reduction of the number of internalized KSHV DNA copies. The above cells were infected with KSHV for 2 h at 37°C, washed, and cultured for another 22 h, and the MOIs were 10 and 5 for HUVECs and SLK cells, respectively. Cellular genomic DNA was extracted, and internalized KSHV DNA was quantitated by amplification of the LANA gene by qRT-PCR. (e, f) The mRNA expression of KSHV genes is down-regulated by AR inhibition. Total RNA from the above HUVECs (e) and SLK cells (f) was subjected to qRT-PCR to determine the expression of LANA, RTA, and PAN. The data were normalized to the actin expression and then calibrated by the control siRNA treatment. (g, h) Knockdown of AR and EphA2 expression decreases nuclear LANA expression in SLK cells, as determined by an immunofluorescence assay and quantitative analysis by Image J software. The infection time was 48 h in this group. Scale bars represent 10 μm. The randomly selected fifty cells in each image were analyzed and the corrected total cell fluorescence (CTCF) of individual images was calculated as following: CTCF = Integrated Density–(Area of selected cells X Mean fluorescence of background readings). Representative images are shown. Data are shown as the mean±SEM; n = 3. Paired Student’s <i>t</i>-tests and one-way ANOVA analysis was respectively performed on (b, c, e and f) and (d, h). * p,0.05, ** p,0.01, ^n.s. p, no significance.