Figure 2.
ZAP-70 contributes to cell survival but is not essential for IgM-stimulated BCR signaling. (A) Strategy for siRNA knockdown of ZAP-70 in primary ZAP-70pos CLL cells and coculture on feeder cells. Downstream analyses were performed on purified CLL cells in monoculture. Created with BioRender.com. (B) MFI of cell surface IgM on CD19+ CLL cells. Primary ZAP-70pos CLL cells (n = 5) were monocultured or cocultured on stromal cells for 1 or 2 days before assessment of surface IgM by fluorescence-activated cell sorting analysis. (C) MFI of cell surface IgM on CD19+ CLL cells transfected with nonspecific siRNA or ZAP-70 siRNA. Primary ZAP-70pos CLL cells (n = 6) were transfected with siRNA and cocultured with stromal cells for 7 days and then separated from stromal cells for an additional 24 hours in monoculture. (D) Representative kinetics plot shows the calcium flux of 1 primary CLL sample after transfection with NSC siRNA or ZAP-70 siRNA. CLL cells were harvested and labeled with Fluo-4 (FITC) after 24 hours in monoculture. Anti-IgM activation was triggered 30 seconds after flow cytometric measurement started. (E) Quantification of calcium flux response of CLL samples (n = 7) transfected with NSC siRNA or ZAP-70 siRNA. Ratios were calculated by using kinetics plots, dividing peak Fluo-4 (FITC-A) intensity induced by anti-IgM activation by baseline Fluo-4 intensity. (F) Representative phospho-BLNK (Tyr96), phospho-Syk (Tyr525/526), phospho-PLCγ2 (Tyr759), total BLNK, total Syk, total PLCγ2, ZAP-70, and β-actin immunoblots of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. To induce BCR signaling, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (G) Quantification of phosphokinases, relative to total (unphosphorylated) proteins after 24 hours in monoculture post-siRNA transfection, using ImageJ software (n = 6). (H) Representative phospho-AKT (s473), phospho-AKT (T308), total AKT, and β-actin immunoblots of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. For anti-IgM–stimulated samples, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (I) Quantification of phospho-AKT relative to total (unphosphorylated) AKT, after 24 hours in monoculture after siRNA transfection, using ImageJ software (n = 4 or 5, as indicated). (J) Percentage of live (Annexin V−, DAPI−) CLL cells, cultured for 48 or 72 hours in monoculture posttransfection (n = 10 and n = 8, respectively). (K) Percentage of live (Annexin V−, DAPI−) CLL cells cultured in monoculture posttransfection (n = 7) and treated with anti-IgM for 48 hours (n = 6). **P < .01. ns, not significant (P > .05); SE, short exposure.

ZAP-70 contributes to cell survival but is not essential for IgM-stimulated BCR signaling. (A) Strategy for siRNA knockdown of ZAP-70 in primary ZAP-70pos CLL cells and coculture on feeder cells. Downstream analyses were performed on purified CLL cells in monoculture. Created with BioRender.com. (B) MFI of cell surface IgM on CD19+ CLL cells. Primary ZAP-70pos CLL cells (n = 5) were monocultured or cocultured on stromal cells for 1 or 2 days before assessment of surface IgM by fluorescence-activated cell sorting analysis. (C) MFI of cell surface IgM on CD19+ CLL cells transfected with nonspecific siRNA or ZAP-70 siRNA. Primary ZAP-70pos CLL cells (n = 6) were transfected with siRNA and cocultured with stromal cells for 7 days and then separated from stromal cells for an additional 24 hours in monoculture. (D) Representative kinetics plot shows the calcium flux of 1 primary CLL sample after transfection with NSC siRNA or ZAP-70 siRNA. CLL cells were harvested and labeled with Fluo-4 (FITC) after 24 hours in monoculture. Anti-IgM activation was triggered 30 seconds after flow cytometric measurement started. (E) Quantification of calcium flux response of CLL samples (n = 7) transfected with NSC siRNA or ZAP-70 siRNA. Ratios were calculated by using kinetics plots, dividing peak Fluo-4 (FITC-A) intensity induced by anti-IgM activation by baseline Fluo-4 intensity. (F) Representative phospho-BLNK (Tyr96), phospho-Syk (Tyr525/526), phospho-PLCγ2 (Tyr759), total BLNK, total Syk, total PLCγ2, ZAP-70, and β-actin immunoblots of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. To induce BCR signaling, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (G) Quantification of phosphokinases, relative to total (unphosphorylated) proteins after 24 hours in monoculture post-siRNA transfection, using ImageJ software (n = 6). (H) Representative phospho-AKT (s473), phospho-AKT (T308), total AKT, and β-actin immunoblots of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. For anti-IgM–stimulated samples, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (I) Quantification of phospho-AKT relative to total (unphosphorylated) AKT, after 24 hours in monoculture after siRNA transfection, using ImageJ software (n = 4 or 5, as indicated). (J) Percentage of live (Annexin V−, DAPI−) CLL cells, cultured for 48 or 72 hours in monoculture posttransfection (n = 10 and n = 8, respectively). (K) Percentage of live (Annexin V−, DAPI−) CLL cells cultured in monoculture posttransfection (n = 7) and treated with anti-IgM for 48 hours (n = 6). **P < .01. ns, not significant (P > .05); SE, short exposure.

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