Fig 2: MafA-Dependent Nicotinic Receptor Expression Modulates Insulin Secretion(A–H) Immunohistochemistry of MafAWT and MafARIP islets to show expression of CHRNA4 (A and B; green), CHRNA5 (C and D; green), CHRNB2 (E and F; green), and CHRNB4 (G and H; green). ß cells are stained for insulin (red) and nuclei (DAPI; blue); scale bar represents 20 µm.(I) qPCR amplification of Chrn upstream sequences after immunoprecipitation of ßTC-6 chromatin with a MAFA or rabbit IgG antibody are presented as % input. Differences in percent input for IgG reflect variations in primer efficiencies. n = 4.(J) Induction of luciferase reporter activity of ChrnB2 (pB2LUC) and ChrnB4 (pB4LUC) luciferase reporter constructs upon co-transfection with MAFA. Empty vector control (pGl2b and pFOX) is set to one; n = 3 or 4.(K) qPCR measurements of Chrn expression levels in MafA siRNA-treated ßTC6 cells; n = 3. Data were normalized to the geomean of HPRT and ß-actin mRNA levels.(L) Dynamic insulin secretion of MafAWT and MafARIP islets stimulated with 10 mM glucose (G) and 100 µM nicotine (NIC), 100 µM nicotine + 100 µM oxotremorine (NIC+OXO), and 100 µM oxotremorine (OXO). The transient decrease in insulin secretion upon NIC treatment in MafAWT islets is marked by a solid arrow. The biphasic insulin secretion induced by NIC+OXO treatment is indicated by a dashed arrow and a dotted arrow; n = 8.(M) Dynamic insulin secretion of wild-type islets with 1 or 10 µM acetylcholine (n = 5). Acetylcholine treatment is illustrated by black lines.Data are mean ± SEM and were analyzed using one-way ANOVA and Tukey multiple comparison tests (J) or paired t test (I and K). *p < 0.05 and **p < 0.01. See Figure S2B for validation of the nicotinic receptor antibodies.

Fig 3: Neurotransmitter receptor expression in embryonic pancreas. (A-P) Immunohistochemical analysis of neurotransmitter receptor expression in wild-type and MafB-/- E18.5 pancreas. Co-staining for insulin (green) and glucagon (white) with neurotransmitter receptor proteins in red – ChrnA3 (A,B), ChrnA4 (C,D), ChrnB2 (E,F), ChrnB4 (G,H), Adra2A (I,J), P2ry1 (K,L), Robo1 (M,N) and PlxnA3 (O,P) – visualized using a Zeiss LSM780. Nuclei are stained in blue. Arrows mark insulin producing cells co-expressing respective neurotransmitter receptor proteins, arrowheads denote PlxnA3+ cells in (P), insets show co-expression of neurotransmitter receptor proteins with insulin and glucagon. Scale bar: 20 µm.