Apoptosis induced by IL-21 is dependent on c-Myc. (A) Wild-type RC-K8 and IL-21–resistant RC-K8R cells were treated with IL-21 (100 ng/mL) for 6 hours and RNA was collected as specified in “Microarray hybridization and analysis.” Microarray gene expression analysis was performed. Presented is a heat map of 42 genes with the most dramatic changes between RC-K8 and RC-K8R upon IL-21 treatment. (B) RC-K8, MC116, RC-K8R, and OCI-LY-3 cell lines and cells from a primary DLBCL tumor from patient 4 were treated with IL-21 (100 ng/mL). At 24, 48, and 72 hours after treatment, cellular proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted for c-Myc. Immunoblotting for GAPDH served as a loading control. The cell line results are representative of 3 independent experiments. Normalized densitometry of c-Myc/GAPDH ratio is shown. The values in specimens at time point 0 were arbitrarily defined as 1. *A statistically significant difference (P < .05) between experimental conditions marked by arrowheads. (C) RC-K8 and MC-116 cells were transfected with siRNA targeting c-Myc or control siRNA. Twenty-four hours after transfection, cells were treated with IL-21 (100 ng/mL) for 24 hours. Cellular proteins from untreated and IL-21–treated cells were resolved by SDS-PAGE and immunoblotted for c-Myc or GAPDH. (D) RC-K8 cells were transfected with shRNA targeting c-Myc or control shRNA. Twenty-four hours after transfection, cells were treated with IL-21 (100 ng/mL) for 24 hours. Cellular proteins from untreated and IL-21–treated cells were resolved by SDS-PAGE and immunoblotted for c-Myc or GAPDH. All immunoblots shown in panel D originated from the same membrane and identical exposure; superfluous lanes were removed. Cell viability in panels C-D was assayed by YO-PRO/PI staining after 48 hours of IL-21 treatment. The results in panels C-D are representative of 3 independent experiments.