Induction of Hb switching and RBC apoptosis by xTRAIL1. (A) Cross-linked FLAG-xTRAIL1 (100 or 200 ng) or an anti-FLAG antibody as a control (200 ng) was injected into the tail vein of 3 tadpoles for each condition at stage 58 (top panels). His-tagged xDR-M1-LBR-His (400 ng) or His peptide (400 ng) as a control was injected into the tail vein of 3 tadpoles for each condition at stage 59 (middle panels) and into the peritoneal cavity of 3 tadpoles at stage 64 (bottom panels). RBCs were isolated 1 week (top panels), 18 days (middle panels), and 22 days (bottom panels) after injection. The lysates of the RBCs were then analyzed by SDS-PAGE and stained with Coomassie brilliant blue (CBB) (shown at left). The ratio of the band intensity corresponding to adult-type Hb to the sum of the band intensities corresponding to adult- and larval-type Hbs was calculated for each sample. Right-hand panels show the mean and SD from the 3 individual animals used in each experiment; **P < .01 and *P < .05 compared with control. (B) RBCs isolated from a tadpole at stage 57 or from an adult frog were treated with cross-linked xTRAIL1 (20 ng/mL). After 24 hours, the cells were observed by phase contrast microscopy. Scale bars, 20 μm. (C) RBCs were treated with cross-linked FLAG-xTRAIL1 (top panel) and xDR-M1-LBR-His (bottom panel). After 24 hours, the cell viability was quantified by the CellTiter-Glo luminescent cell viability assay. Cell viability is shown as the percentage of the value obtained using xTRAIL1-untreated cells. The data represent the mean (n = 3) and SD; *P < .001 compared with control (top panel); *P < .05 and **P < .01 compared with no treatment with xDR-M1-LBR-His of xTRAIL1 (bottom panel). (D) Isolated RBCs were pretreated with or without Z-VAD-FMK (20 μM) for 1 hour and then were treated with cross-linked xTRAIL1 (25 ng/mL). After 24 hours, the cell viability was measured and shown as described in panel C. The data represent the mean (n = 4) and SD; *P < .01 compared with no treatment with Z-VAD-FMK of xTRAIL1.