Fig. 3

MiR-493-5p negatively regulated the differentiation of Th9 cells in vitro. The CD4⁺T cells were separated from widetype mice by density gradient centrifugation and magnetic beads. To investigate the effect of miR-493-5p on Th9 cells differentiation, both CD4⁺T cells which were transfected with miR-493-5p mimic or treated with the inhibitor and their negative control (NC) were under the condition of inducing Th9 cell differentiation. A–C The mRNA level of IL-9, IRF4 and FOXO1 was detected by RT-qPCR. The data showed that the mRNA of FOXO1, IL-9 and IRF4 were downregulated in CD4⁺T cells treated with miR-493-5p mimic and upregulated in CD4⁺T cells treated with miR-493-5p inhibitor. D Groups. E–H The protein production of IL-9, IRF4 and FOXO1 was detected by western blot. The data showed that protein of FOXO1, IL-9 and IRF4 were downregulated in CD4⁺T cells treated with miR-493-5p mimic and upregulated in CD4⁺T cells treated with miR-493-5p inhibitor. I, J The proportion of CD4⁺Th9 cells was analysed by flow cytometry and K IL-9 secretion in cell supernatant was measured by ELISA, and the data were all consistent with the proportion of Th9 cells. Data are from three experiments (mean and SD of three independent replicates).^**P < 0.01 compared to A group, ^#P < 0.05 compared to B group, ^##P < 0.01 compared to B group