Notch1

After transfection of miRNA-122 mimic and miRNA-122 inhibitor groups, the total protein of HUVECs cells was extracted by adding RIPA buffer (Vazyme Biotech, China). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane by polyacrylamide sulfate gel electrophoresis. After the transfer, the membrane was sealed with 5% Bowene Serum Albumin at room temperature for 1h and subsequently incubated overnight with the required primary antibody at 4°C. The next day, the membrane was incubated with Sangon Biotech (China, 1:500 dilution) for 1h, and the protein was observed by enhanced chemiluminescence (Vazyme Biotech, China). G:BOX Chemi XX9 imaging system (Syngene, UK) for protein visualization was used. Individual western blotting was performed with primary antibody, Bax, Bcl-2, Caspase-3 antibody (Abcam, USA, 1:1000 diluted), Hes1, Notch1, Vascular Endothelial Growth Factors [VEGF], CCNG1 and GAPDH antibody (Sangon Biotech, China, 1:500 diluted).

Total RNA was isolated from ADMSCs using the TRIzol (Invitrogen, USA) extraction method. cDNA libraries were synthesized from extracted mRNA using the Reverse Transcription Reagent kit (Takara, Japan) and random primers, while the cDNA library of miRNA was synthesized using the miRcute Plus miRNA First-Strand cDNA synthesis Kit (TIANGEN, China). Samples were submitted for Real-Time PCR using SYBR Green mix (Takara, Japan), and results were normalized to Gapdh (for mRNA) or U6 (for miRNA) expression levels. Gapdh, Cd31, Vegf, and Notch-1 specific primers were obtained from Sangon Biological Engineering (Shanghai, China), while miR-1956, miR-192-3p, miR-138-5p, and U6 specific primers were obtained from RiboBio. Primer sequences are presented in Table S2. Data were analyzed through the comparative Ct (^ΔΔCt) method to quantify relative gene expression.