Fig. 5. The Notch1 signalling pathway regulated the GABAB1 receptor via hes1.
A–D Hes1 expression changes following regulation of the Notch1 signalling pathway in the mPFC of MIP mice by Two-way ANOVA followed by LSD test. Hes1 mRNA [A main effect of AAV, F (1, 20) = 17.47, P < 0.001; METH, F (1, 20) = 2.89, P > 0.05; AAV × METH, P > 0.05] and protein [C main effect of AAV, F (1, 20) = 25.73, P < 0.05; METH, F (1, 20) = 3.16, P > 0.05; AAV × METH, P > 0.05] levels were increased in the syn-NICD-OE group of mice. Hes1 mRNA [B, main effect of AAV, F (1, 20) = 14.17, P < 0.01; METH, F(1, 20) = 12.56, P < 0.01; AAV × METH, P > 0.05] and protein [D, main effect of AAV, F (1, 20) = 11.84, P < 0.01; METH, F (1, 20) = 11.27, P < 0.01; AAV × METH, P > 0.05] levels were decreased in the syn-NICD-shRNA group of mice. E, F Hes1 mRNA (t12 = 2.35, *P < 0.05) and protein (t10 = 2.95, *P < 0.05) levels were significantly lower in syn-Hes1-shRNA mice than control mice by student’s t test. GABAB1 receptor mRNA (t11 = −2.27, *P < 0.05) and protein (t10 = −3.71, **P < 0.01) levels were increased in the syn-Hes1-shRNA group by student’s t test. G, H. The results of ChIP-qPCR assays indicated that Hes1 bound to the promoter of the GABAB1 receptor. G Schematic diagram and table of the GABAB1 promoter region showing potential binding sites of Hes1 predicted by the JASPAR database. H The naive mice mPFC were subjected to ChIP assay. Three paired primers were designed near the predicted binding sites of Hes1. ChIP-qPCR assays verified the association of Hes1 and the promoter of the GABAB1 gene (Primer 3, t10 = −6.11, ***P < 0.001) by student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control saline group. #P < 0.05, ###P < 0.001 vs. paired METH group. The results are expressed as the mean ± SEM, n = 6–7.