Fig. 6

Inhibition of METTL3 activity reduces Foxp3 mRNA stability and expression by decreasing m⁶A modification. a Left: Foxp3 protein expression was determined by Western blot in CD4⁺ T cells treated with either DMSO or STM2457 when cultured under Treg differentiation conditions. BRD4 and MGMT were used as positive controls, and β-actin was used as a loading reference; right: quantification of protein bands, n = 4. b RT-qPCR of METTL3 and Foxp3 mRNA in flow-sorted CD4⁺CD25⁺CD127⁻ cells after treatment with either DMSO or STM2457 when cultured under Treg differentiation conditions. c Top: predicted m⁶A modification sites in the CDS of Foxp3; bottom: meRIP-qPCR (m⁶A immunoprecipitation-qPCR) comparing the m⁶A enrichment on three predicted m⁶A sites of Foxp3 mRNA in DMSO- or STM2457-treated CD4⁺ T cells. The results are presented as a relative percentage normalized to inputs, n = 5. d Degradation of Foxp3 mRNA was measured in differentiated Treg cells treated with either DMSO or STM2457 after the addition of actinomycin D for 0, 2, and 6 h. The results were normalized to 0 h, n = 4. e RT-qPCR of Foxp3 mRNA in CD4⁺ T cells of SLE patients and HCs, n = 24. f Correlation analysis of the mRNA expression of METTL3 and Foxp3 in SLE CD4⁺ T cells, n = 40. g meRIP-qPCR of m⁶A enrichment at three predicted m⁶A sites in Foxp3 mRNA in CD4⁺ T cells isolated from HC, patients with active SLE, and patients with inactive SLE. Inactive SLE: SLEDAI ≤ 4, active SLE: SLEDAI > 4. The results are presented as relative percentages normalized to inputs. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance, two-way ANOVA with Sidak’s multiple comparisons test for a, c and d, unpaired two-tailed Student’s t test for b and e, Pearson’s correlation analysis for f, and Chi-square test for g)