Analysis of bioactivity and mechanisms of direct apoptosis of αCD20-IL-21 fusokine. For A and B, cells were treated with αCD20-IL-21 (1 µg/mL) or equimolar doses of IL-21 or αCD20-IgG1 and immunoblotted with indicated Abs. (C) RCK8 cells were first transfected with siRNA targeting STAT3, Bax, cMyc, or control siRNA, followed by treatment with αCD20-IL-21 (1 µg/mL) or equimolar doses of IL-21 or αCD20-IgG1 at 24 hours after transfection. Percentage of cell death was determined by YO-PRO/PI staining at 72 hours posttreatment, using flow cytometry. Immunoblotting was carried out at 24 hours posttransfection to confirm proteins knockdown. (D) RCK8 cells treated with αCD20-IL-21 (1 µg/mL) or equimolar doses of IL-21 or αCD20-IgG1 were subjected to TUNEL assay via immunofluorescence or (E) flow cytometry. Blue represents DAPI staining and green TUNEL staining; magnification, 20×. TUNEL positivity is indicative of fragmented DNA, a characteristic of apoptotic cells. (F) RCK8 cells were cotransfected with STAT3 luciferase reporter plasmid (pLucTKS3) and internal control plasmid (pRL-TK). At 24 hours posttransfection, cells were exposed to αCD20-IL-21 fusokine or IL-21 for 48 hours, followed by dual luciferase measurements using luminometer. Data represents the luciferase activity normalized to pRL-TK signal. (G) RCK8 cells exposed to αCD20-IL-21 fusokine or IL-21 were stained with YO-PRO-1/PI to measure cell death. EC50 curves were derived by plotting percentage of dead cells, using Prism software. Data are mean ± SD. *P < .05; **P < .01; ***P < .001.