Fig. 7

NeuroD1 is crucial on Ex-4 activating the Wnt/β-catenin signalling to promote the Ins2-encoding brain insulin. The protein levels of NeuroD1 in the hippocampus of HF-diabetic mice A and db/db mice B were detected by Western blotting. Additionally, the mRNA levels of Neurod1 in the hippocampus of HF-diabetic mice C and db/db mice D were measured by RT-qPCR assay. E–G and J–K HT22 cells were treated as in Fig. 1C, And NeuroD1 level was demonstrated by immunoblot in (E). mRNA level of Neurod1 F detected by RT-qPCR assay. G Immunofluorescence was performed for NeuroD1 (green) and DAPI (blue). Scale bar = 100 μm. H Immunoblot with its gray density and I mRNA levels measured by RT-qPCR demonstrated the NeuroD1 changes in HT22 cells treated as in Fig. 6D–H. J The binding sites of NeuroD1 on the Ins2 promoter shown in the diagrammatic drawing, and the K ChIP-qPCR results of the relative binding levels of IgG or NeuroD1 to the input measured by the indicated primers are shown under the diagrammatic drawing. L HT22 cells were transfected with the NeuroD1 expression plasmid and the wild-type Ins2 luciferase reporter construct or its mutation plasmids (MUT1/MUT2). Then, they were incubated in HG or HG + Ex-4 as above. Luciferase activity was measured and normalized to the Renilla control. β-actin was the internal control. Data are presented as the mean ± SD. For A and C, n = 5, ^###P < 0.001. vs. CTL; ^††P < 0.01, ^†††P < 0.001. vs. HF-diabetic. ^‡‡P < 0.001, ^‡‡‡P < 0.001. vs. Ex-4. For B (n = 4) and D (n = 3), ^§§P < 0.01, ^§§§§P < 0.0001 vs. db/db; || P < 0.01. vs. Ex-4. And for E–I (n = 4), J–K (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001