PARP1 is cleaved and caspase-10 is the most active caspase in 11kDa/NS1 transfection– and B19V infection–induced apoptosis. (A-B) Detection of cleaved PARP1 and cleaved caspase-10. CD36⁺ EPCs were infected with B19V, and capsid⁺ cells were sorted at 48 hours after infection by flow cytometer. UT7/Epo-S1 cells were transfected with pGFP control, pGFP-11kDa, and pGFP-NS1. At 48 hours after transfection, the GFP⁺ populations of transfected cells were sorted by flow cytometer. (A) Sorted cells were used for detecting the cleaved PARP1 by Western blot using anti–cleaved PARP1 at a dilution of 1:1000 (Cell Signaling). The blots were reprobed with anti–β-actin. (B) Sorted cells were used for detecting of active caspase-10 by Western blot using anti–caspase-10 at a dilution of 1:1000 (Sigma-Aldrich). Uninfected or pGFP-transfected cells served as mock as shown. The blots were reprobed with anti–β-actin. (C) Detection of activated caspase-3&7, -6, -8, -9, and -10 in 11kDa- and NS1-transfected cells. UT7/Epo-S1 cells were transfected with pRFPHA control, pRFP-11kDaHA, and pRFP-NS1HA. FAM-labeled FLICA peptides, FAM-DEVD-FMK, FAM-VEID-FMK, FAM-LETD-FMK, FAM-LEHD-FMK, and FAM-AEVD-FMK were used to detect active caspase-3/7, caspase-6, caspase-8, caspase-9, and caspase-10, respectively. Individual FLICA staining was performed to determine active caspases at 48 hours after transfection. Transfected cells were selected by intracellular staining of anti-HA tag at 1:100 dilution (clone HA-7; Sigma-Aldrich), shown as HA⁺ and HA⁻, and were plotted to FLICA signal detected by flow cytometer. (D) Detection of activated caspase-3&7, -6, -8, -9, and -10 in B19V-infected CD36⁺ EPCs. CD36⁺ EPCs were infected with B19V. At 48 hours after infection, cells were used for individual FLICA staining followed by intracellular staining with the antibody against B19V capsid. Both capsid-positive and -negative cells were plotted to FLICA signal detected by flow cytometer. TX indicates transfection.