Fig. 6

Map3k9 is the target gene of miR-20a to suppress Treg differentiation in EAE. A The expression of Map3k9 was upregulated in CD4⁺ T cells with miR-20a knockdown. B Sequences of WT and mutant 3’ UTR of Map3k9 and miR-20a mimics used in the luciferase assays. C Relative luciferase activity of the reporter carrying the WT or mutant Map3k9 3’ UTR or NC 3’ UTR co-transfected with miR-20a mimics or NC mimics into HEK-293 cells. The activity of Renilla luciferase was used to eliminate variances between groups. D, E Flow cytometric analysis of the CD4⁺ T cells transfected with NC or miR-20a antagomirs, or miR-20a antagomirs + Map3k9 siRNA after stimulated with MOG_35-55 peptide in Treg-polarizing condition for 5 days. The percentages of CD25⁺Foxp3⁺ cells are shown in the CD4⁺ gate. The flow cytometry plots shown are representative of 4 independent experiments. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 using unpaired (A, C) and paired (E) Student’s t-test