Fig 2: The p.Ser64Phe (S64F) mutation greatly stabilizes MAFA in human EndoC-ßH1 cells grown in 1.1 or 15.5 mM glucose. (A) EndoC-ßH1 cells were transfected with wild type (WT) and p.Ser64Phe (S64F) MAFA-Myc and, after 48 h, incubated with medium containing 1.1 mM or 15.5 mM glucose for an additional 12 h. The transfected cells were then incubated with 25 µg/mL cycloheximide (CHX) for the indicated time. Transfected MAFA-Myc and endogenous ß-actin protein levels were determined by immunoblotting (IB) using anti-Myc and anti–ß-actin antibodies, respectively. (B) The Myc protein band intensity was measured in the 15.5 mM glucose sample, normalized to ß-actin, and plotted as a percentage of the initial band intensity. (C) No significant difference was found between WT and p.Ser64Phe (S64F) MAFA mRNA levels in transfected cells grown in 1.1 mM glucose. Endogenous MAFA mRNA levels also did not change under these conditions. Student’s two-tailed t test. n.s., not significant. n = 3. Error bars represent SEM.

Fig 3: The mobility of p.Ser64Phe (S64F) MAFA is indistinguishable from the p.Ser65Ala (S65A) kinase mutant. (A) Schematic of MAFA showing sites of phosphorylation (red dots) within the transactivation, DNA-binding (basic), and dimerization region (leucine zipper, L-zip). (B) Wild type (WT) and mutant MAFA transfected HeLa nuclear extracts were incubated at 37 °C for 40 or 80 min (40' or 80') in the presence of the phosphatase inhibitor, sodium orthovanadate (Na3VO4, 10 mM), or NaCl (10 mM). The arrowheads denote the location of fully phosphorylated MAFA (F-P, blue), the form lacking Ser65 and GSK3-mediated phosphorylation (Un-P, red), and the completely dephosphorylated protein produced by incubating in the presence of NaCl (De-P, white).

Fig 4: Islet-like clusters express specific markers of beta cells and secrete insulin in response to glucose challenge. a The mRNA expression profile of components implicated in the commitment, differentiation, and maturation of beta cells was examined in differentiated islet-like cell clusters by RT-PCR. Shown is the expression in 6-week differentiated (Diff) MDSC and in Min6 insulinoma cells as a control. b Protein expression profiles were also probed by Western blot analysis at three different time points,: undifferentiated PP8 MDSC (MDSC), early differentiated MDSC (Diff-Ea: PP8 + 2 weeks differentiation), and late differentiated MDSC (Diff-La: PP8 + 6 weeks differentiation). Shown are typical profiles blotted for Nestin, Glut2, Pdx1, Nkx2-2, and MafA. Alpha-Tubulin is shown last as a loading control. Molecular weight markers are indicated in kDa. c Glucose-responsive insulin secretion: for each assay, 4 to 5 islet-like cell clusters differentiated for 8–10 weeks were collected in 24-well dishes and starved in KREBS buffer for 2 h before incubating for 1 h in KREBS supplemented with 2.8 mM glucose and subsequently incubated for 1 h in 20 mM glucose. Secreted insulin was measured by FRET. Whereas little or no insulin was released in 2.8 mM glucose, when further challenged with 20 mM glucose, clusters secreted significant levels of insulin with regard to the small number of cells in the wells (1500 to 2000 cells/well, n = 3); *P < 0.01

Fig 5: Immunofluorescence analysis of beta cell markers in islet-like cell clusters differentiated from MDSC. a Islet-like clusters were differentiated in vitro for 6 weeks from MDSC derived from nestin-eGFP transgenic mice. Photomicrographs show staining for eGFP and insulin (red) and DNA (blue). The bottom panels show a merged view of GFP, insulin, and DNA (left panel) and the phase-contrast image of the cluster with staining for insulin and DNA (right panel). Scale bar = 50 µm. b–d MDSC differentiated into islet-like clusters for 6 weeks were dissociated by short enzymatic treatment and collected by cytospin centrifugation, before fixation and staining for the expression of transcription factors implicated in beta-cell differentiation and insulin. Shown are fluorescence micrographs of dissociated cells stained for DNA and Nkx2.2 or DNA and Pax6 (b), co-stained for PDX1 and insulin (c), and for Nkx6.1 and MafA (d). Scale bars = (b) 20 µm, (c,d) 10 µm