Up-regulation of CD46 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for CD46 expression. Top- Percentage of CD46 positive cells for each MSC population. Bottom- MFI ratio for CD46 expression on each MSC population. MFI ratio  =  (Median Fluorescence Intensity for CD46/Median Fluorescence Intensity for isotype control). The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. 0(B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD46 and unfilled histograms are the staining with the corresponding isotype control.</p

Effect of overexpression of HCMV US2 protein on MSC in a complement-mediated cytotoxicity assay.

<p>(A) The results depict representative data from a flow cytometry-based complement-mediated cytotoxicity assay from at least 3 independent experiments. The assay was performed in the presence of human IgM and rabbit serum complement (experimental) or in the presence of human IgM and absence of the rabbit serum complement (control). MSC stained with calcein were counted as viable, cells stained with Ethidium homodimer-1 were counted as dead, and cells doubly positive were counted as having membrane damage. Doubly negative cells were excluded from the analysis. A live/dead control (top) was used in order to set up the quadrant gates for alive, dead, and membrane-damaged populations and to set parameters for compensation between the 2 fluorescent channels. The experimental assay provided the percentage of dead cells under experimental conditions. The control assay provided the percentage of spontaneously dead cells. The same conditions were applied for non-transduced MSC and MSC-US2. (B) The percentage of cytotoxicity for each cell line was calculated as follows: (percent of dead cells under experimental conditions – percent of spontaneously dead cells)/(100 – spontaneously dead)*100. The percentage of experimental or spontaneously dead cells was determined by calculating the percentage of cells that were positive for Ethidium homodimer-1, and negative for calcein. The results depict the mean ± SEM of three independent experiments. * indicates p<0.05.</p

Up-regulation of CD59 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for expression of CD59. Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD59, and unfilled histograms are the corresponding isotype control staining. The MFI ratio for each MSC cell line was obtained by performing the following calculation: MFI ratio  =  (Median Fluorescence Intensity for CD59/Median Fluorescence Intensity for isotype control). (B) MFI ratio for each MSC population is shown. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC.</p

Up-regulation of CD55 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed for CD55 surface expression by flow cytometry. Top- Percentage of CD55 positive cells for each MSC population. Bottom- MFI ratio for CD55 expression on different MSC populations. MFI ratio  =  Median Fluorescence Intensity for CD55/Median Fluorescence Intensity for isotype control. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. (B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to each MSC cell population stained with antibody against CD55 and unfilled histograms correspond to isotype staining.</p