Figure 4.
ZAP-70 enhances CCR7 signaling and chemotaxis. (A) Migration capacity in response to chemokine CCL19 or CCL21 (1 μg/mL, 3 hours) were quantified in primary CLL cells (n = 11) transfected with nonspecific siRNA or ZAP-70 siRNA. Percentage of migrated CLL cells were quantified by using counting beads and divided numbers of migrated CD5+ CD19+ cells through transwells by total cell numbers that were loaded onto transwells. Nontreated controls were the samples that were cultured in media without chemokines. (B) Representative fluorescence-activated cell sorter histograms (left) and quantification (right, n = 6) of surface CCR7 levels on CD19+ cells in primary CLL cells transfected with nonspecific siRNA or ZAP-70 siRNA. (C) ZAP-70 immunoblots of whole-cell lysates from MEC1/parental or MEC1/ZAP-70 cells. Cells were treated with doxycyline (1 μg/mL) for 72 hours as indicated. (D) Migration capacity in response to chemokine CCL19 (1 μg/mL, 3 hours) was quantified in MEC1/parental cells or MEC1/ZAP-70 cells treated with 1 μg/mL doxycycline. Percentage of migrated MEC1 cells were quantified using counting beads and numbers of migrated CD19+ cells through transwells were divided by total input cells numbers. (E) Phospho-AKT (T308), phospho-AKT (S473), total AKT, and ZAP-70 immunoblots of primary CLL cells after nonspecific control siRNA or ZAP-70 siRNA transfection. For cytokines stimulated samples, cultured CLL cells were treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes. (F) Graphs of relative phosphorylated AKT level (relative to total AKT levels) in CLL samples transfected with control siRNA or ZAP-70 siRNA and treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes. (G) Phospho-LCP1 (S5), total LCP1, ZAP-70, and β-actin immunoblots of primary CLL cells after nonspecific control siRNA or ZAP-70 siRNA transfection. For cytokines stimulated samples, cultured CLL cells were treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗P < 5 × 10−2; ∗∗P < 10−3. ns, not significant.

ZAP-70 enhances CCR7 signaling and chemotaxis. (A) Migration capacity in response to chemokine CCL19 or CCL21 (1 μg/mL, 3 hours) were quantified in primary CLL cells (n = 11) transfected with nonspecific siRNA or ZAP-70 siRNA. Percentage of migrated CLL cells were quantified by using counting beads and divided numbers of migrated CD5+ CD19+ cells through transwells by total cell numbers that were loaded onto transwells. Nontreated controls were the samples that were cultured in media without chemokines. (B) Representative fluorescence-activated cell sorter histograms (left) and quantification (right, n = 6) of surface CCR7 levels on CD19+ cells in primary CLL cells transfected with nonspecific siRNA or ZAP-70 siRNA. (C) ZAP-70 immunoblots of whole-cell lysates from MEC1/parental or MEC1/ZAP-70 cells. Cells were treated with doxycyline (1 μg/mL) for 72 hours as indicated. (D) Migration capacity in response to chemokine CCL19 (1 μg/mL, 3 hours) was quantified in MEC1/parental cells or MEC1/ZAP-70 cells treated with 1 μg/mL doxycycline. Percentage of migrated MEC1 cells were quantified using counting beads and numbers of migrated CD19+ cells through transwells were divided by total input cells numbers. (E) Phospho-AKT (T308), phospho-AKT (S473), total AKT, and ZAP-70 immunoblots of primary CLL cells after nonspecific control siRNA or ZAP-70 siRNA transfection. For cytokines stimulated samples, cultured CLL cells were treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes. (F) Graphs of relative phosphorylated AKT level (relative to total AKT levels) in CLL samples transfected with control siRNA or ZAP-70 siRNA and treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes. (G) Phospho-LCP1 (S5), total LCP1, ZAP-70, and β-actin immunoblots of primary CLL cells after nonspecific control siRNA or ZAP-70 siRNA transfection. For cytokines stimulated samples, cultured CLL cells were treated with CCL19 or CCL21 at 1 μg/mL for 5 minutes (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗P < 5 × 10−2; ∗∗P < 10−3. ns, not significant.

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