Effect of CIB1 knockdown on productive KSHV trafficking in endothelial cells.

<p>(A) Untransduced or CIB1 sh-RNA transduced HMVEC-d cells were incubated with KSHV (20 DNA copies/cell) and FITC labeled dextran for 30 min at 37°C. After washing, cells were harvested, fixed, permeabilized, and examined by FACS. Unstained HMVEC-d cells were used as negative control. The results are shown in a histogram as a percentage of fluorescent cells with a fluorescence intensity higher than the unstained negative control. The mean fluorescence intensity (MFI) as an indication of dextran uptake was measured and the table shows representative MFI values from three independent experiments. (B) Serum starved control or CIB1- shRNA transduced cells were infected with KSHV (20 DNA copies/cell) for 10 min, washed, and processed for double immunofluorescence staining using mouse anti-KSHV glycoprotein gpK8.1A and rabbit anti-Rab5 antibodies, followed by anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 antibodies, respectively. Representative deconvoluted images are shown. Boxed areas from the merged panel are enlarged in the rightmost panel and arrows indicate KSHV particles in proximity to Rab5 positive vesicles (green).