Figure 2
Figure 2. IL-3 induces Pim1 protein expression in basophils. (A) Effects of immunoregulatory and proinflammatory cytokines on basophil apoptosis. Basophils were cultured for 24 hours in the absence or presence of the indicated cytokines. Activation of caspase 3–dependent cascade evidenced by procaspase 3, PARP, and Bcl-2 proteolytic cleavage was determined by immunoblotting. Cytokines were used at the following concentrations: 20 ng/mL (IL-3, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and TSLP), 50 ng/mL (IL-5, GM-CSF, and NGF). (B,C) IL-3 supports basophil survival and induces Pim1 protein expression under serum-free and serum-containing culture conditions. (B) Basophils were cultured in serum-free medium (RPMI supplemented with 1 mg/mL of BSA) or in serum-containing medium (RPMI supplemented with 10% FCS) for 24 hours in the presence of increasing concentrations of IL-3 (0-50 ng/mL) as indicated. Apoptosis was evaluated by annexin V–FITC staining. Data are from 3 independent experiments and presented as the mean plus or minus SEM. *P < .05 for comparison of no FCS versus 10% FCS. †P < .05 for IL-3 treatment versus medium control, by ANOVA. (C) Dose-response of Pim1 induction by IL-3. Pim1, procaspase 3, activated caspase 3, as well as phosphorylation status of Stat5 were analyzed by Western blotting using specific antibodies. β-Actin is shown as loading control. (D) Time-course analysis of Pim1 protein expression induced in basophils by IL-3. Basophils were incubated in the presence of IL-3 (50 ng/mL) for indicated time periods. Pim1 protein expression was analyzed by immunoblotting. (E) Comparison of the effects of priming cytokines on Pim1 expression in basophils. Basophils were cultured for 24 hours with IL-3, IL-5, GM-CSF, or NGF (all at 50 ng/mL) before analysis of Pim1 by Western blotting. (F) Pim1 is induced in human granulocytes on stimulation with prosurvival cytokines of the GM-CSF family. Freshly isolated granulocytes (Fr.is.) or cells cultured for 24 hours in medium alone or in the presence of the indicated cytokine (all at 50 ng/mL). See also Pim1 expression in neutrophils and eosinophils cultured with all members of the GM-CSF family in Figure S2.

IL-3 induces Pim1 protein expression in basophils. (A) Effects of immunoregulatory and proinflammatory cytokines on basophil apoptosis. Basophils were cultured for 24 hours in the absence or presence of the indicated cytokines. Activation of caspase 3–dependent cascade evidenced by procaspase 3, PARP, and Bcl-2 proteolytic cleavage was determined by immunoblotting. Cytokines were used at the following concentrations: 20 ng/mL (IL-3, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and TSLP), 50 ng/mL (IL-5, GM-CSF, and NGF). (B,C) IL-3 supports basophil survival and induces Pim1 protein expression under serum-free and serum-containing culture conditions. (B) Basophils were cultured in serum-free medium (RPMI supplemented with 1 mg/mL of BSA) or in serum-containing medium (RPMI supplemented with 10% FCS) for 24 hours in the presence of increasing concentrations of IL-3 (0-50 ng/mL) as indicated. Apoptosis was evaluated by annexin V–FITC staining. Data are from 3 independent experiments and presented as the mean plus or minus SEM. *P < .05 for comparison of no FCS versus 10% FCS. †P < .05 for IL-3 treatment versus medium control, by ANOVA. (C) Dose-response of Pim1 induction by IL-3. Pim1, procaspase 3, activated caspase 3, as well as phosphorylation status of Stat5 were analyzed by Western blotting using specific antibodies. β-Actin is shown as loading control. (D) Time-course analysis of Pim1 protein expression induced in basophils by IL-3. Basophils were incubated in the presence of IL-3 (50 ng/mL) for indicated time periods. Pim1 protein expression was analyzed by immunoblotting. (E) Comparison of the effects of priming cytokines on Pim1 expression in basophils. Basophils were cultured for 24 hours with IL-3, IL-5, GM-CSF, or NGF (all at 50 ng/mL) before analysis of Pim1 by Western blotting. (F) Pim1 is induced in human granulocytes on stimulation with prosurvival cytokines of the GM-CSF family. Freshly isolated granulocytes (Fr.is.) or cells cultured for 24 hours in medium alone or in the presence of the indicated cytokine (all at 50 ng/mL). See also Pim1 expression in neutrophils and eosinophils cultured with all members of the GM-CSF family in Figure S2.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal