Fig 2: Effect of Notch blockade on signaling parameters.Representative H&E (A), Alcian blue (B), and Ki-67 (C) staining of uninfected (N), CR-infected (CR), or CR + DBZ-treated (CR + DBZ) Rag1−/− mice (scale bar 100 μm; 7–8 mice/group; n = 3 independent experiments). D Total crypt RNA from the same group of mice was isolated and Notch1 expression was investigated via real-time RT-qPCR. The values were normalized to GAPDH and data presented as fold change. P values = 0.000004 (Notch1), 0.000091 (Hes1); n = 3 independent experiments. E Western blot analysis of NICD in the isolated crypts in the indicated group. β-Actin was used as the loading control. Lower panel. Densitometry showing relative abundance. *,**P < 0.05; n = 3 independent experiments. F Real-time RT-qPCR of Notch downstream target Hes-1 in the isolated crypts from the indicated groups. P values = 0.000091 (Hes1); n = 3 independent experiments. Lower panel showing western blot of Hes-1 and Math-1 in the crypts isolated from the indicated groups. Lamin B was used as the loading control (n = 3 independent experiments).

Fig 3: MDK knock down inhibits MDK-related signaling. siCon and siMDK were transfected with H1299 and A549 cells, and then cell migration and signaling were measured by immunoblotting assays after exposure to normoxic or hypoxic conditions. (A) H1299 and A549 cells were plated in serum-free medium onto the upper chamber, and 10% FBS medium was contained in the bottom chamber to act as a chemoattractant; the cells were incubated at 37 °C in a CO2 incubator for 16 h. Student’s t test was performed and the values displayed as average ±SD; n = 6, * p < 0.05. (B) Identification of MDK-Notch2-Hes-1 signaling. (C) Checking the PI3K-AKT signaling. (D) Examining the EMT-related proteins. (E) Checking anti-apoptotic and pro-apoptotic proteins.

Fig 4: Effect of miR-375 on the Notch signaling pathway. (A) Luciferase reporter gene assay verification of the bioinformatics analysis-predicted NOTCH2 as a target gene of miR-375, **P<0.01. (B) NOTCH2, Delta-like 1 (DLL1) and hes family bHLH transcription factor 1 (HES1) mRNA expression levels detected by reverse transcription-quantitative polymerase chain reaction and normalized to GAPDH. Data are the mean ± standard deviation (SD) of three experiments, *P<0.05 and **P<0.01. (C) Western blotting demonstrating the inhibitory effect of miR-375 on the Notch signaling pathway. (D) Relative protein expression levels. Data are the mean ± SD of three experiments, *P<0.05 and **P<0.01.

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.