Comparison of apoptosis induced by transfection of 3 B19V nonstructural proteins and by B19V infection. (A-C) Comparison of apoptosis induced by 7.5kDa, 11kDa, and NS1 in UT7/Epo-S1 cells. UT7/Epo-S1cells were transfected with pGFP control, pGFP-7.5kDa, pGFP-11kDa, and pGFP-NS1. Cells were analyzed at the 3 time points after transfection as indicated. (A) The absolute percentages of annexin V⁺ populations of GFP⁺ cells were subtracted from those of GFP⁻ cells and are plotted to the time points after transfection as shown. (B) UT7/Epo-S1 cells were transfected with plasmids expressing GFP or GFP-fused proteins as indicated. The mean fluorescence intensity (MFI) of GFP or GFP-fused proteins was detected by flow cytometer and plotted at 3 time points after transfection. (C) The absolute values shown in panel A were normalized by the MFI of GFP shown in panel B that serves as a marker of protein expression level. The normalized data were plotted as relative values to GFP-11kDa, arbitrarily set as 100%. (D-F) Comparison of apoptosis induced by 7.5kDa, 11kDa, and NS1 in CD36⁺ EPCs. The same plasmids, as indicated, were transfected to CD36⁺ EPCs. (D) The absolute percentages of annexin V⁺ populations of GFP⁺ cells were subtracted from those of GFP⁻ cells and are plotted to the time points after transfection as shown. (E) CD36⁺ EPCs were transfected with plasmids expressing GFP or GFP-fused proteins as indicated. The MFI of GFP or GFP-fused proteins was detected by flow cytometer and plotted at 24 hours after transfection. (F) Results shown in panel D were normalized by following the same method used in panel C. (G) Apoptosis induced during B19V infection of CD36⁺ EPCs. The extent of apoptosis induced by mock/B19V infection of CD36⁺ EPCs was detected by TUNEL assay. Cells were also immunostained at the time points as indicated with an anti-B19V capsid antibody (clone 521-5D; Millipore) at 1:100 dilution followed by a fluorescein isothiocyanate–conjugated secondary antibody with the TUNEL assay simultaneously. Stained cells were analyzed by flow cytometer, and both capsid⁺ and capsid⁻ cell populations of B19V-infected cells were gated for TUNEL⁺ population. TX indicates transfection.