Geneglobe program

To screen for angiogenic markers, we used RT² Profiler™ PCR Array Human Angiogenesis (Cat#: PAHS-024Z, Qiagen, Germantown, MD, USA) and RT² Profiler™ PCR Array Human Notch Signaling Pathway Plus (Cat#: PAHS-059Y). Each array contains 86 genes, 5 housekeeping genes, human genomic DNA contamination, reverse transcription, and positive PCR controls. Total RNA from HUVECs (control and treated) was extracted using an RNA extraction kit (Qiagen). A total of 0.5 µg of mRNA was used for cDNA synthesis using the RT² First Strand Kit (Cat# 330401, Qiagen). cDNA was then mixed with 2 × RT2 SYBR Green qPCR Master Mix and RNase- and DNase-free water and loaded onto the plates following the manufacturer’s instructions. RT-PCR was performed with a CFX96 thermocycler (Bio-Rad, Hercules, CA, USA) All the samples passed the quality control checks using the GeneGlobe Program provided by Qiagen (https://geneglobe.qiagen.com/us/analyze, accessed on 30 January 2023). RT2 PCR array data were normalized against the housekeeping genes (GAPDH, ACTB, B2M, RPLP0, and HPRT1) by calculating the 2^−∆∆CT for each gene of interest in the plate. Heatmaps were generated and analyzed by using GeneGlobe Program. Finally, the candidate genes were chosen to be validated in an additional qRT-PCR experiment.