Transfection of 11kDa induces apoptosis in both B19V permissive and nonpermissive cells. (A-B) UT7/Epo-S1 cells, CD36⁺ EPCs, HeLa cells, K562 cells, and 293 cells were transfected with pGFP-11kDa plasmid (A) or pGFP as a control (B). CD36⁺ EPCs were stained at 24 hours after transfection; other cells were stained at 48 hours after transfection with annexin V/PI double staining, followed by flow cytometric analysis. Both GFP-negative (GFP⁻) and GFP-positive (GFP⁺) cell populations were gated to plot cells by PI versus annexin V. Only a representative experiment is shown, and the percentage of each quadrant is indicated. Annexin V⁺ population is a combination of the annexin V⁺/PI⁺ population (number in the upper right quadrant) with the annexin V⁺/PI⁻ population (number in the lower right quadrant). (C) The experiments as described in panels A and B were performed at least 3 times independently. The percentage value of annexin V⁺/PI⁺ or annexin V⁺/PI⁻, as shown in individual panel with indicated cell type, was calculated by subtracting the annexin V⁺/PI⁺ population or the annexin V⁺/PI⁻ population of GFP⁺ cells by that of GFP⁻ cells (background apoptosis). (D) UT7/Epo-S1 cells were transfected with pGFP-11kDa and stained with DAPI. Confocal images were taken at a magnification of 60× (objective lens) with an Eclipse C1 Plus confocal microscope (Nikon). Arrows show apoptotic nuclei, which were enclosed in the apoptotic bodies visualized by GFP fluorescence. Results that are shown as average ± SD in all the figures are generated from at least 3 independent experiments. Tx indicates transfection.