IL-21–induced apoptosis is dependent on STAT-3 and Bax and rescued by Bcl-2 and Bcl-X_L. (A) RC-K8 cells were transfected with PCDNA3.1–BCL-XL, –BCL-2, –MCL-1, or empty vector. Seventy-two hours after transfection, cells were treated with IL-21 (100 ng/mL). After 24 hours, cellular proteins from untreated and IL-21–treated cells were resolved by SDS-PAGE and immunoblotted for Bcl-X_L, Bcl-2, Mcl-1, or GAPDH. Cell viability was assayed by YO-PRO/PI staining after 48 hours of IL-21 treatment. (B) RC-K8 cells were transfected with siRNA targeting Bax or control siRNA. Twenty-four hours after transfection, cells were treated with IL-21 (100 ng/mL). Cellular proteins from untreated and IL-21–treated cells were resolved by SDS-PAGE and immunoblotted for Bax and c-Myc at 24 hours after treatment. Immunoblotting for GAPDH served as a loading control. Cell viability was assayed by YO-PRO/PI staining after 48 hours of IL-21 treatment. (C-D) RC-K8 cells were transfected with siRNA targeting STAT3 or control siRNA. Seventy-two hours after transfection, cells were treated with IL-21 (100 ng/mL). Cellular proteins from untreated and IL-21–treated cells were resolved by SDS-PAGE and immunoblotted for p-STAT3 and STAT3 at 15 minutes after treatment (C) and for c-Myc, Bcl-X_L, Bcl-2, and STAT3 at 24 hours after treatment (D). Cell viability was assayed by YO-PRO/PI staining after 48 hours of IL-21 treatment (C). The results in panels A through D are representative of 3 independent experiments. (E) A model of IL-21–induced apoptosis in DLBCL. IL-21 binding to IL-21Rα results in Jak activation and subsequent phosphorylation and activation of STAT3. Homodimerized STAT3 enters the nucleus and activates transcription of c-Myc. c-Myc protein promotes the transcription of Bax and suppresses Bcl-2 and Bcl-X_L, thus disrupting the Bcl-2 rheostat within the cells and triggering mitochondrial outer membrane permeabilization, caspase-9 activation, and apoptosis.