Fig 2: Western blot protein analysis of cells excised individually from striatal and hippocampal tissues. (A) The SDS-PAGE blots show the expression of dopamine D2 receptor (Drd2) and a4-nicotinic acetylcholine receptor (a4-nAChR) in the striatum (top) and hippocampus (bottom). (B) Results from the quantitative analysis of Drd2 and a4-nAChR expression (mean ± SEM; expressed as ratio to GAPDH) in the striatum (top) and hippocampus (bottom). Striatal and Drd2 and a4-nAChR protein expressions were significantly increased relative to sham control group. Significant increases in Drd2 and a4-nAChR protein expressions in the hippocampus also were found. *, **, and *** indicate significant protein expression with P < 0.05, P < 0.01, and P < 0.001, respectively, relative to sham control group.

Fig 3: The relative effect of specific α7-nAChR antagonists to block ACh (10−5 M) elicited responses.A- αBgTx attenuated the ACh evoked contractions in a concentration dependent manner. The blocking effects of 50 nM and 100 nM toxin were statistically significant. The recovery is almost complete. B- The block by 50 nM αA-CTx PIVA was not significant, while the α-CTx ImI at 100 nM blocked almost completely the ACh elicited contraction. The recovery from block was partial. n = number of muscles. Asterisks indicate a significant difference from the control value: *P<0.01.

Fig 4: 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). β 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 βTC-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 βTC6 cells; n ≥ 3. Data were normalized to the geomean of HPRT and β-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 5: 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.