Fig. 5: TEPP-46 inhibits Smad2/3 activation during T_H17 differentiation.

(A) Schematic showing cytokines that drive RORγT⁺ T_H17 and FoxP3⁺ T_reg cell differentiation. (B) T_H17 cells skewed for 24 hours with TEPP-46 (50μM) or vehicle (DMSO) were lysed and cell extracts subjected to immunoblot analysis of phosphorylated STAT3 (P-STAT3) and total STAT3 (T-STAT3). Quantification is pooled densitometric analysis from n=7 mice from three independent experiments. Paired Student’s t-test. (C) Flow cytometry analysis of TGF-β1-mediated induction of FoxP3 in the presence of indicated concentration of TEPP-46 or DASA-58 or vehicle (DMSO). Quantification is of n=6–7 mice per group pooled from N=3 independent experiments. Repeated measures one-way ANOVA with Tukey post test. (D) assessment of P-SMAD2 in response to TGF-β1 treatment in Jurkat cells pretreated with DMSO or TEPP-46. Quantification of N=4, each dot pair represents one independent experiment; paired t test. (E) Cells treated with TGF-β1 in the presence of TEPP-46 (50μM) or vehicle (DMSO) for 24 hours were subjected to qRT-PCR analysis for Ccr8 and Irf8 expression. Quantification is of n=6 mice per group pooled from N=3 independent experiments. Repeated measures one-way ANOVA with Tukey post test. (F) Purified CD4⁺ T cells were skewed towards T_H17 in the presence of increasing concentrations of TGF-β1 in the presence of TEPP-46 (50μM) or vehicle (DMSO) for 48 hours and subjected to flow cytometry analysis of IL-17A and GM-CSF production after 6 hours of PMA/ionomycin stimulation. Quantification is of n=4 mice per group pooled from N=2 independent experiments. Repeated measures two-way ANOVA with Tukey post test. **p<0.01, ****p<0.0001.