Figure 1.
ZAP-70 contributes to the constitutive phosphorylation of AKT and GSK3β in IGHV–unmutated CLL cells. (A) Representative kinetic blots show the calcium flux of IGHV–unmutated (UM-CLL) or IGHV–mutated (M-CLL) CLL cells with NSC or ZAP-70 siRNA transfection. CLL cells were harvested and labeled with Fluo-4 (fluorescein isothiocyanate). BCR-signaling was activated with anti-IgM beads, triggered 20 to 30 seconds after flow cytometric measurement started. (B) Quantification of calcium flux response of CLL samples (n = 4 for both UM-CLL and M-CLL) transfected with NSC or ZAP-70 siRNA. The Ca2+ ratios were calculated by using kinetic blots and divided peak Fluo-4 (FITC-A) mean intensity upon anti-IgM activation by baseline. (C) Phospho-AKT (T308), phospho-AKT (S473), total AKT, ZAP-70, and β-actin immunoblots of primary M-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM stimulated samples, CLL cells were treated with beads-bound anti-IgM for 20 minutes. (D) Phosphorylated protein levels of AKT (relative to total AKT levels) 24 hours after siRNA transfection in M-CLL samples. For anti-IgM stimulated samples, monocultured M-CLL cells were treated with beads-bound anti-IgM for 20 minutes (n = 4 for pi-AKT [T-308] and n = 3 for pi-AKT [S473]). (E) Phospho-GSK-3β (S9), total GSK-3β, ZAP-70, and β-actin immunoblots of primary UM-CLL or M-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM stimulated samples, monocultured CLL cells were treated with beads-bound anti-IgM for 20 minutes. (F) Phosphorylated GSK-3β levels (relative to total GSK-3β levels) 24 hours after siRNA transfection in UM-CLL and M-CLL samples. For anti-IgM stimulated samples, monocultured CLL cells were treated with beads-bound anti-IgM for 20 minutes (n = 3 for both UM-CLL and M-CLL). (G) Cell viability of primary CLL cells assessed using annexin-V/propidium iodide staining, 9 days after siRNA transfection. Cells were monocultured for 48 hours after nonspecific control siRNA or ZAP-70 siRNA transfection for 7 days on stromal cells. (H) MCL1 and β-actin immunoblots of primary UM-CLL cells cultured under identical conditions as described in panel G. (I) Relative MCL1 protein level (compared to with β-actin levels) 24 hours after siRNA transfection in UM-CLL samples (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗P < 5 × 10−2, ∗∗∗P < 10−4. LE, long exposure; ns, not significant; NSC, non-specific control; SE, short exposure.

ZAP-70 contributes to the constitutive phosphorylation of AKT and GSK3β in IGHV–unmutated CLL cells. (A) Representative kinetic blots show the calcium flux of IGHV–unmutated (UM-CLL) or IGHV–mutated (M-CLL) CLL cells with NSC or ZAP-70 siRNA transfection. CLL cells were harvested and labeled with Fluo-4 (fluorescein isothiocyanate). BCR-signaling was activated with anti-IgM beads, triggered 20 to 30 seconds after flow cytometric measurement started. (B) Quantification of calcium flux response of CLL samples (n = 4 for both UM-CLL and M-CLL) transfected with NSC or ZAP-70 siRNA. The Ca2+ ratios were calculated by using kinetic blots and divided peak Fluo-4 (FITC-A) mean intensity upon anti-IgM activation by baseline. (C) Phospho-AKT (T308), phospho-AKT (S473), total AKT, ZAP-70, and β-actin immunoblots of primary M-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM stimulated samples, CLL cells were treated with beads-bound anti-IgM for 20 minutes. (D) Phosphorylated protein levels of AKT (relative to total AKT levels) 24 hours after siRNA transfection in M-CLL samples. For anti-IgM stimulated samples, monocultured M-CLL cells were treated with beads-bound anti-IgM for 20 minutes (n = 4 for pi-AKT [T-308] and n = 3 for pi-AKT [S473]). (E) Phospho-GSK-3β (S9), total GSK-3β, ZAP-70, and β-actin immunoblots of primary UM-CLL or M-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM stimulated samples, monocultured CLL cells were treated with beads-bound anti-IgM for 20 minutes. (F) Phosphorylated GSK-3β levels (relative to total GSK-3β levels) 24 hours after siRNA transfection in UM-CLL and M-CLL samples. For anti-IgM stimulated samples, monocultured CLL cells were treated with beads-bound anti-IgM for 20 minutes (n = 3 for both UM-CLL and M-CLL). (G) Cell viability of primary CLL cells assessed using annexin-V/propidium iodide staining, 9 days after siRNA transfection. Cells were monocultured for 48 hours after nonspecific control siRNA or ZAP-70 siRNA transfection for 7 days on stromal cells. (H) MCL1 and β-actin immunoblots of primary UM-CLL cells cultured under identical conditions as described in panel G. (I) Relative MCL1 protein level (compared to with β-actin levels) 24 hours after siRNA transfection in UM-CLL samples (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗P < 5 × 10−2, ∗∗∗P < 10−4. LE, long exposure; ns, not significant; NSC, non-specific control; SE, short exposure.

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