Fig 2: Changes in MafA expression were associated with METTL3-regulated GSIS from NIT-1 cells. (A) Immunoblot of MafA expression in NIT-1 cells transfected with si-METTL3 + pcDNA or si-METTL3 + pcDNA MafA. (B) Quantification of the immunoblot in (A). (C) The GSIS index of the cells described in (A). (D) Immunoblot of MafA expression in NIT-1 cells transfected with Ad-METTL3 + si-NC or Ad-METTL3 + si-MafA and treated with 1 mM MG for 24 h. (E) Quantification of the immunoblot in (D). (F) The GSIS index of the cells described in (D). Results are presented as means ± SD of n = 3‐4 independent experiments. * p < 0.05.

Fig 3: Loss of METTL3 attenuates the expression of MafA. (A–D) METTL3 and MafA protein and mRNA levels were measured by immunoblotting and qPCR, respectively, in NIT-1 cells transfected with METTL3 siRNA (si-METTL3) or a nonspecific control siRNA (si-NC), which was set to 1. (E–G) MafA protein and mRNA expression levels in NIT-1 cells transfected with Ad-METTL3 or a nonspecific control adenovirus (Ad-NC) and treated with 1 mM of MG for 24 h were measured by immunoblotting and qPCR, respectively. (H, I) m6A MafA mRNA levels as detected by MeRIP-qPCR in NIT-1 cells transfected with si-METTL3 or si-NC, which was set to 1 (H), or Ad-METTL3 or Ad-NC and treated with 1 mM of MG for 24 h (I). (J, K) MafA mRNA as measured by qPCR in NIT-1 cells transfected with si-METTL3, si-NC, Ad-METTL3, or Ad-NC and treated with ActD to block transcription. Results are presented as the means ± SD of n = 3–4 independent experiments. * p < 0.05.

Fig 4: MAFA protein levels were transiently upregulated at 4 weeks in male MafAS64F/+ islets(A) Immunostaining over the course of 3, 4, 5, 6, and 7 weeks revealed that MAFA protein staining intensity only increased at 4 weeks of age in male MafAS64F/+ (Het) islets.(B) In contrast, MAFA staining intensity was unchanged between female MafAS64F/+ and WT islets.(C) MafA mRNA levels were significantly reduced at 4 and 5 weeks in both male and female MafAS64F/+ islet samples. Fold change shown relative to male WT islets. Two-tailed Student t test; *p < 0.05; scale bars, 50 μm.

Fig 5: Non–β cell hormone expression is increased in MafA∆β islets.(A and B) Analysis of MafA, Insulin 1 (Ins1), and non–β cell hormone mRNA levels in MafAWT and MafA∆β islets from 3-month-old adult male (A) and female (B) mice. Data are shown as mean ± SEM. n = 3–4 animals/group. *P < 0.05 by Student’s t test; n.d., not detectable. (C) Gast mRNA was induced more in male than in female MafA∆β islets. Data are shown as mean ± SEM. n = 3–4 animals/group. *P < 0.05 by Student’s t test. (D) Top panel: Stomach G cells from GASTWT and GASTKO mice served as positive and negative control for Gast antibody staining. n = 2–3 animals/group. Middle panels: Representative images for Gast (red) and Insulin (white) immunostaining in male MafAWT and MafA∆β islets. Insets for Gast staining are shown in right panels. Bottom left panel: Gastrin signal (A.U.) per mouse islet cell. Bottom right panel: Gast+ cells per islet (% of islet cells). Four to 6 islets per mouse. n = 3–4 animals/group were quantified using ImageJ. Scale bar, 50 μm. Data are shown as mean ± SEM. *P < 0.05 by Student’s t test.