KSHV infection increases the number of PML nuclear bodies and associated proteins in endothelial cells.

(A) HUVECs were either infected with rKSHV.219 at an MOI of 20, or HCMV at an MOI 3, or left uninfected (n.i.). 48 h after infection cells were fixed and processed for IFA staining. Infected cells express GFP, PML was detected with a mouse anti-PML antibody and a goat anti-mouse IgG Lissamine Rhodamine (LRSC)-conjugated secondary antibody and DNA was stained with DAPI. Images were acquired using a Carl Zeiss microscope at 100x magnification. For better visualization PML NBs are shown as black dots on a white background and the nuclei were encircled. Bars, 10 μm. (B) For the quantification of PML NBs upon KSHV and HCMV infection, PML NBs were counted in at least 50 cells per sample and a Mann Whitney U test was performed. Boxes indicate 25th to 75th percentile; the central line inside each box indicates the median and the whisker illustrate the 5th and 95th percentile. The dots indicate the outliers. (C) Protein expression of infection markers and PML NBs-associated proteins was analyzed by WB 48 h post infection of HUVECs infected with either KSHV or HCMV. (D) HEK-293 cells were treated with IFN-α for 72 h and PML protein expression was analyzed by WB.