Trizol solution

Total RNAs were extracted using the Trizol solution (Qiagen, USA). Then a total of 1 μg RNA was subjected to reverse transcription reactions using a Reverse Transcription kit (Qiagen, USA). PCR amplification was conducted using the SYBR GREEN qPCR mix (Thermo, USA).
PD-L1 forward primer: 5’- TGGCATTTGCTGAACGCATTT-3’,
PD-L1 reverse primer: 5’-TGCAGCCAGGTCTAATTGTTTT-3’;
METTL16 forward primer: 5’-CTCTGACGTGTACTCTCCTAAGG -3’,
METTL16 reverse primer: 5’- TACCAGCCATTCAAGGTTGCT-3’.

The TRIzol solution (Qiagen, Switzerland) was used to extract the total RNA from the HUVECs according to the instructions of the manufacturer, followed by transcribing the RNAs to cDNA using the SuperScript III (Takara, Tokyo, Japan) reagent. The SYBR green quantitative PCR kit (Takara, Tokyo, Japan) was used to determine the relative expression of target genes, while GAPDH was taken as the negative control. QRT-PCR was performed on the ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems, CA, USA). The 2^-ΔΔCt method was used to calculate and quantify the relative expression level of target genes. The following primers were used in this study: LOX-1 (F: 5’-TGATAGAAACCCTTGCTCG-3’, R: 5’- TTGCTTGCTGGATGAAGTC-3’); MCP-1 (F: 5’-GTTGGCTCAGCCAGATGCA-3’, R: 5’-AGCCTACTCATTGGGATCATCTTG-3’); CXCL-2 F: 5’-GGCAGAAAGCTTGTCTCAACCC-3’, R: 5’-CTCCTTCAGGAACAGCCACCAA-3’); eNOS (F: 5’-GTGGCTGTCTGCATGGACCT-3’, R: 5’-CCACGATGGTGACTTTGGCT-3’); Occludin (F: 5’-TACAGCAATGGAAAACCACACT-3’, R: 5’-CAAAGGAATGGGAAACGACTAA-3’); KLF2 (F: 5’-CCAAGAGTTCGCATCTGAAGGC-3’, R: 5’-CCGTGTGCTTTCGGTAGTGGC-3’); GAPDH (F: 5’-GACGGCCGCATCTTCTTGT-3’, R: 5’-CAGTGCCAGCCTCGTCCCGTAGA-3’).

Total RNA extraction from hUC-MSCs was performed using the Trizol solution (Qiagen, Valencia, CA, USA) and cDNA was obtained from total RNA using an Advantage RT-PCR kit (Clontech, Palo Alto, CA, USA). Table 1 shows the primer sequences used for real-time qPCR. The following genes were examined: Plexin A4 (PLXNA4), Formin 1 (FMN1), Amphiregulin (AREG), Stathmin 2 (STMN2), Serpin Family I Member 1 (SERPINI1), Microtubule-associated protein 2 (MAP2), Neuronal Differentiation 1 (NEUROD1), Glial fibrillary acidic protein (GFAP), Myelin basic protein (MBP), and Neurofilament-L (NF-L).
Real-time qPCR was performed according to the SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA), which enables real-time quantitative detection of PCR products based on SYBR green fluorescence due to the incorporation of SYBR green into double-stranded DNA. The results were analyzed by a comparative cycle threshold (CT) method for the quantification of relative gene expression for the housekeeping gene (GAPDH).