IL-21 mediates its biologic effects through activation of STAT3. (A) Serum-starved MWCL-1 or freshly sorted CD19⁺CD138⁺ WM tumor cells (n = 3) were stimulated with 100 ng/mL IL-21 for 10 minutes. After fixation and permeabilization, tyrosine phosphorylation of STATs 1, 3, 4, 5, and 6 was determined via FACS analysis. Shown is a representative example of 3 separate experiments. Gray histograms indicate baseline STAT phosphorylation, with open histograms representing expression of the phosphorylated isoform. (B) Immunoblot analysis of tyrosine phosphorylation of STATs 1, 3, and 5 in serum-starved MWCL-1 cells treated with 100 ng/mL IL-21 for the indicated times. Total STAT1, STAT3, and STAT5 were used as loading controls. Shown are representative blots of 3 separate experiments. (C) Inhibition of IL-21 activity through the use of the STAT3 inhibitor, STATTIC. MWCL-1 cells were pretreated for 20 minutes in the presence or absence of 0.5μM STATTIC before the addition of 100 ng/mL IL-21 where indicated. Cells were cultured for 72 hours, and proliferation, IgM secretion, and viability were assessed as outlined in “Methods.” Each experiment was performed 3 times, and data represent the mean ± SD. *Statistically significant at P < .05. Immunoblotting for pSTAT3 was performed on MWCL-1 cells pretreated in the presence or absence of 0.5μM STATTIC for 20 minutes, followed by stimulation with 100 ng/mL IL-21 for 10 minutes. Cells were lysed in RIPA buffer and SDS-PAGE was performed. After probing for pSTAT3, blots were stripped, and total STAT3 was used as a loading control.