Additional file 6 of m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus
Creators: name: Lu, Shuang
name: Wei, Xingyu
name: Zhu, Huan
name: Hu, Zhi
name: Zheng, Meiling
name: Wu, Jiali
name: Zhao, Cheng
name: Yang, Shuang
name: Feng, Delong
name: Jia, Sujie
name: Zhao, Hongjun
name: Zhao, Ming
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 6: Figure S1. METTL3 expression varies after T-cell activation and Treg-cell differentiation in vitro. a Representative dot plots showing the proportions of CD4+ T cells before and after activation by anti-CD3 and anti-CD28 antibodies for 3 days. b The mRNA expression of METTL3 in CD4+ T cells activated by anti-CD3 and anti-CD28 antibodies at 0, 12, 48, and 72 h was detected by RT-qPCR, n = 3. c Left: Western blot of METTL3 expression in Th0 and differentiated Tregs at 3 and 5 days, and β-actin was used as a loading control; right: quantification of METTL3 protein expression in Th0 and differentiated Tregs, n = 3. (***p &lt; 0.001, ns, no significance, one-way ANOVA with Dunnett’s multiple comparisons test for b, two-way ANOVA with Sidak’s multiple comparisons test for c). Figure S2. Anti-SRBC antibodies and splenic Tfh cells are significantly elevated in SRBC-immunized mice. a ELISA of anti-SRBC specific antibodies IgM, IgG1, IgG2a, IgG2b, IgG (H+L), and IgG3 in the blood serum of control mice and SRBC-immunized mice. Serum dilution: IgM: 1:200; IgG1, IgG2a, and IgG (H+L): 1:400; IgG2b: 1:800; IgG3: 1:40; n = 6. b Left: representative dot plots showing the proportion of splenic CD4+ T cells in mice after SRBC challenge; right: quantification of splenic CD4+PD1+CXCR5+ Tfh cells, n = 6. (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 ****p &lt; 0.0001, unpaired two-tailed Student’s t test). Figure S3. Proportions of Tfh and Th2 cells remain comparable between control and STM2457-administered cGVHD mice. a Comparison of the body weights of cGVHD mice body treated with DMSO or STM2457. b Left: spleen and dLN images of cGVHD mice; right: quantification of spleen weight. c Top: representative dot plots showing the proportion of CD4+ T cells in the spleen and dLNs of cGVHD mice treated with DMSO or STM2457; bottom: quantification of CD4+PD1+CXCR5+ Tfh cells. d Left: representative dot plots showing the proportion of splenic CD4+ T cells in the spleens of cGVHD mice treated with DMSO or STM2457; right: quantification of CD4+IL-4+ Th2 cells. e ELISA of IFN-γ and IL-17A levels in the blood serum of cGVHD mice treated with DMSO or STM2457. (ns, no significance, unpaired two-tailed Student’s t test). Figure S4. METTL3 catalytic inhibition or gene knockdown suppresses CD4+ T-cell activation in vitro. a Comparison of total m6A modification between STM2457- and DMSO-treated CD4+ T cells by colorimetric quantification, n = 5. b Left: representative dot plots showing the composition of CD4+ T cells in STM2457 (5 μM)-treated and DMSO control cells after activation by anti-CD3 and anti-CD28 antibodies for 3 days; right: quantification of nonactivated CD4+CD25−CD69− cells, n = 5. c Immunoblot of METTL3 in CD4+ T cells treated with either METTL3 siRNA or scramble siRNA, and β-actin was used as a loading control. d Left: representative dot plots showing the proportion of CD4+ T cells treated with either scramble siRNA or METTL3 siRNA for 3 days after being activated by anti-CD3 and anti-CD28 antibodies; right: quantification of unactivated CD4+CD25−CD69− cells, n = 3. e Left: representative dot plots showing the proportion of CD4+ T cells treated with either DMSO or STM2457 for 5 days when cultured under Treg differentiation conditions; right: quantification of CD4+CD25+CD127− cells, n = 4. f The mRNA expression of METTL3 in SLE CD4+ T cells treated with either DMSO or STM2457 when cultured under Treg differentiation conditions was detected by RT-qPCR, n = 4. (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ns, no significance, unpaired two-tailed Student’s t test).