Figure 2.

Overexpression of IRS1 reduces Treg cell differentiation. (A–E) Naïve CD4 T cells isolated from C57BL/6J mice were cultured for 1 day under Th0 conditions and then transduced with the MIEG3-IRS1 vector. After transduction, cells were cultured for 2 days under Treg-inducing conditions. (A) FOXP3 protein levels were measured via flow cytometry. (B–D) GFP+ cells, sorted using a flow cytometer, were used. (B) Irs1 and Foxp3 mRNA levels were measured via qRT-PCR. (D) Y612-phosphorylated form and whole protein of IRS1 were measured via Western blot analysis. β-actin was used as an internal control. (E) After transduction, PBS or IGF1 (100 ng/mL) or insulin (100 ng/mL) were treated and cultured for 2 days under Treg conditions. FOXP3 expression was measured via flow cytometry. (F) Naïve CD4 T cells were transduced with the MIEG3-IRS1 vector (as in (A)). The cells were then differentiated into each subset for 2 days. IFN-γ, IL-13, and IL-17 proteins were measured via flow cytometry. Expression was measured via qRT-PCR and normalized to that of Gapdh. All data are representative of three independent experiments. The bar graph next to the flow cytometry plot shows data pooled from three independent experiments. The error bars represent the SD, and (A–C,F) p-values were calculated using a t-test. (E) p-value of the bottom graph was calculated using one-way ANOVA/Tukey test. * p < 0.05, ** p < 0.01, **** p < 0.0001. ns: not significant.