IL-21 regulates gene and protein expression in WM. (A) qRT-PCR analysis of transcription factors involved in B-cell differentiation. MWCL-1 cells were treated with 100 ng/mL IL-21 for 48 hours, at which time mRNA was extracted and qRT-PCR was performed. Data are presented as the fold change in expression relative to untreated MWCL-1, with a value of 1 indicating no change in transcript expression on treatment with IL-21. Representative data from 1 of 3 separate experiments are shown. (B) Immunoblot analysis of transcription factors involved in B-cell differentiation. MWCL-1 cells were treated with increasing concentrations of IL-21 (0-200 ng/mL) for 72 hours, at which time cells were lysed with RIPA buffer and SDS-PAGE was performed. After probing for specific transcription factors, blots were stripped and reprobed for actin, which was used as a loading control. Shown are representative blots of 3 separate experiments. (C-D) ELISA was performed for (C) IL-10 or (D) IL-6 with the use of cell-free supernatants collected from MWCL-1 cells cultured in IL-21 for 72 hours. Each experiment was performed in triplicate on 3 separate occasions, and data represent the mean ± SD. (E) Freshly sorted CD19⁺CD138⁺ WM tumor cells (n = 4; WM26-28, WM19) were cultured with or without IL-21 for 72 hours. Cell-free supernatants were collected, and the concentration of IL-6 was determined by ELISA. Data represent the mean ± SD of a single experiment performed in triplicate. *Statistically significant at P < .05.