Fig 2: Notch-induced glucose intolerance is only partially reversible.(A) Experimental schematic used for Dox withdrawal (Dox-off). (B) Notch target (left) and β cell maturity gene (right) expression in islets isolated from 8 weeks’ Dox-off β-tetO-NICD and Cre– mice (n = 4 mice/group). (C) Representative images of pancreatic sections from 8 weeks’ Dox-off β-tetO-NICD and Cre– mice, with quantitation of β cell nuclear Mafa fluorescence intensity (n = 5 mice/group). Scale bars: 20 μm. (D) GTT in male and (E) female Dox-off β-tetO-NICD and Cre– controls (n = 7–9 mice/group). AUC, area under the curve (mg × dL/min). (F) Serum insulin after oral glucose challenge in Dox-off β-tetO-NICD and Cre– controls (n = 5 mice/group). (G) Glucose-stimulated insulin secretion (GSIS) in islets isolated from Dox-off β-tetO-NICD and Cre– mice, adjusted for islet insulin content (n = 5 mice/group). All data are shown with group means; *, P < 0.05, **, P < 0.01, ***, P < 0.001 by 2-tailed t test.

Fig 3: MAFB is expressed in both human alpha and beta cells.(A) Different antibodies were tested for their specificity toward mouse MafB by triple immunofluorescent (IF) staining of mouse pancreatic sections. Representative images of triple IF staining with anti-glucagon, insulin, and hMAFA, mMafB1, hMAFB2 and hMAFB3 antibodies. Note that hMAFB3 did not show an alpha cell specific pattern as did mMafB1 and hMAFB2 antibodies. (B) MAFB expression was detected in both human pancreatic alpha and beta cells. Representative images of triple IF-staining showing MAFB expression in human islets from healthy individuals. The percentages of positive cells for MAFB in glucagon+ (green) and insulin+ (red) cells were 67.2±7.6% MAFB+ alpha cells and 53.6±10.5% MAFB+ beta cells. Results are the averaged expression ± S.E.M of counting results from n = 4 control individuals (463 GLU+ cells and 797 INS+ cells were counted in total). Scale bar = 25 µM. (C) MAFB expression in insulin+ PDX1+ cells. The percentage of double insulin+ PDX1+ cells positive for MAFB was 30.2±5.8% (858 INS+ PDX1+ counted cells in total). The above percentages of expression are the averaged counting results ±S.E.M from n = 3 control individuals. Scale bar = 25 µM.

Fig 4: Glucagon, insulin and MAFA immunohistochemistry of case 1 (IV4). Immunohistochemical staining of serial sections of the resected pancreatic specimen of case 1 showing a solitary insulinoma (left panels (a,c,e)) and normal pancreatic tissue with multiple pancreatic islets (right panels (b,d,f)).

Fig 5: Nrf2 knockdown blocks the anti-oxidative effects and β-cell function restoration by hUC-MSCs under high-glucose conditions. (A) INS-1E cells were seeded into Transwell 6-well plates and treated with scrambled or Nrf2 siRNA. Then, INS-1E cells cultured with or without hUC-MSCs were exposed to high-glucose (30 mM) medium or normal medium for 24 h. (B) Nrf2 siRNA knockdown was confirmed by western blotting and quantitative real-time PCR. (C) Apoptosis of INS-1E cells was analysed by flow cytometry. (D) Intracellular ROS were assessed by flow cytometry. (E) GSIS in INS-1E cells was measured at 1 h after glucose stimulation (16.7 mM) by ELISA. (F and G) Protein levels of Nrf2, HO-1 (F), PDX-1, GLUT2, MafA, BCL-2, and cleaved Caspase-3 (G) in INS-1E cells were measured by western blotting. Protein expression levels were normalized to GAPDH. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.