American Society of Hematology

Figure 4

$Figure 4. Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells. (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells (Ctrl). Graph shows mean + SEM of 3 independent experiments. Ikaros expression was also determined by Western blot. Anti–mouse β-actin antibodies were used to control loading. (B) In vivo binding of Ikaros to the il2 promoter in anergic Th1 cells was determined by ChIP using anti-Ikaros antibodies and primers for the il2 promoter on ionomycin-treated (Iono) and control resting (Rest) Th1 cells. ChIPs were also performed with a control IgG. PCR products are shown for precipitated fractions and sample inputs. One experiment of 4 with similar results is shown. Numbers below input bands represent relative amount (to control cells) quantified using qPCR. Reactions were carried out with 3 different amounts of sample (2-fold increases) to confirm linearity of the amplification. Graph shows relative band (to control cells) intensity of immunoprecipitated complexes from all experiments (mean + SEM). (C) CD4+ T cells from DO11.10 mice were isolated from OVA-fed mice and age- and sex-matched PBS-fed controls. Anergy was assessed by ELISA following stimulation with anti-CD3 and anti-CD28. Bars show mean + SEM of 3 different experiments. Chromatin complexes were immunoprecipitated using an anti-Ikaros antibody. PCR products using primers for the il2 promoter are shown for the precipitated fraction and the sample inputs. Gel shows one experiment of 4 with similar results. Numbers below input bands represent relative amount (to the value in PBS-fed mice) quantified using qPCR. Reactions were carried out with different amounts of immunoprecipitated sample (2-fold increases) to confirm linearity of the amplification. Graph shows relative intensity of immunoprecipitated complexes from 4 different sets of mice (mean + SEM). *P < .02 when comparing band intensities from PBS- and OVA-fed mice using a t test. (D) Samples from panel B were analyzed using primers for mouse actin. (E) ChIP assays were performed using anti–mouse HDAC2 antibodies on samples prepared from resting (Rest) Th1 cells or cells treated with ionomycin (Iono) ± TSA. Samples were amplified with primers for the il2 promoter. Gel shows 1 of 3 experiments. Numbers below input bands represent relative amount (to the value in resting cells) quantified using qPCR.$

Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells. (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells (Ctrl). Graph shows mean + SEM of 3 independent experiments. Ikaros expression was also determined by Western blot. Anti–mouse β-actin antibodies were used to control loading. (B) In vivo binding of Ikaros to the il2 promoter in anergic Th1 cells was determined by ChIP using anti-Ikaros antibodies and primers for the il2 promoter on ionomycin-treated (Iono) and control resting (Rest) Th1 cells. ChIPs were also performed with a control IgG. PCR products are shown for precipitated fractions and sample inputs. One experiment of 4 with similar results is shown. Numbers below input bands represent relative amount (to control cells) quantified using qPCR. Reactions were carried out with 3 different amounts of sample (2-fold increases) to confirm linearity of the amplification. Graph shows relative band (to control cells) intensity of immunoprecipitated complexes from all experiments (mean + SEM). (C) CD4⁺ T cells from DO11.10 mice were isolated from OVA-fed mice and age- and sex-matched PBS-fed controls. Anergy was assessed by ELISA following stimulation with anti-CD3 and anti-CD28. Bars show mean + SEM of 3 different experiments. Chromatin complexes were immunoprecipitated using an anti-Ikaros antibody. PCR products using primers for the il2 promoter are shown for the precipitated fraction and the sample inputs. Gel shows one experiment of 4 with similar results. Numbers below input bands represent relative amount (to the value in PBS-fed mice) quantified using qPCR. Reactions were carried out with different amounts of immunoprecipitated sample (2-fold increases) to confirm linearity of the amplification. Graph shows relative intensity of immunoprecipitated complexes from 4 different sets of mice (mean + SEM). *P < .02 when comparing band intensities from PBS- and OVA-fed mice using a t test. (D) Samples from panel B were analyzed using primers for mouse actin. (E) ChIP assays were performed using anti–mouse HDAC2 antibodies on samples prepared from resting (Rest) Th1 cells or cells treated with ionomycin (Iono) ± TSA. Samples were amplified with primers for the il2 promoter. Gel shows 1 of 3 experiments. Numbers below input bands represent relative amount (to the value in resting cells) quantified using qPCR.

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