Exploitation of the Complement System by Oncogenic Kaposi's Sarcoma-Associated Herpesvirus for Cell Survival and Persistent Infection
Figure 2
Detection and quantification of C5b-9 and C3b depositions on latently KSHV-infected endothelial cells.
(A) Detection of C5b-9 deposition on TIME-KSHV but not TIME cells. Cells were incubated with 10% normal or heat-inactivated human serum for 30 min and stained for C5b-9 deposition by immunofluorescence staining. The scale bar is 50 µm. (B) Quantification of C5b-9 deposition on TIME and TIME KSHV cells. Ten images from each treatment shown in Figure S2 were analyzed for C5b-9-positive areas using the ImageJ software. The average C5b-9 staining area per cell was calculated by dividing the total pixels of C5b-9 staining in a microscopic field by the cell number. Each dot represents the analyzed value from one field. The average values of ten analyses were indicated as horizontal black bars. *** P<0.001. (C) Flow cytometry analysis of C3b staining on TIME and TIME-KSHV cells. White areas are C3b-positive cells. Grey areas are cells stained with an isotype-matched antibody control. (D) Cell surface localization of C5b-9 deposition on TIME-KSHV cells. TIME-KSHV cells were incubated with 10% normal human serum for 30 min, and stained for C5b-9 (red), integrin αVβ3 (captured in the far-red channel but pseudo-colored in green to facilitate visualization) to label plasma membrane and DAPI (blue) to localize the nucleus. Z-stack images were acquired with confocal laser-scanning microscopy. Three-dimensional software was used to generate z-projection images from at least 70 confocal images of 0.1 µm sections. The 3-D images (XY panels) were rotated on the x-axis (XZ panels) and y-axis (YZ panels) to visualize C5b-9 localization on the cell membrane. Arrows show representative areas of C5b-9 depositions on cell surfaces. Images of TIME cells incubated with normal human serum, and TIME and TIME-KSHV cells incubated with heat-inactivated serum are shown in Figure S3.