Thermo scientific revertaid

The total RNA was extracted from HUVECs using an RNA extraction kit (RN07; Aidlab, Beijing China), according to the manufacturer's instructions. This was then reverse transcribed into cDNA using the Thermo Scientific RevertAid (K1622; Thermo Fisher Scientific, Waltham, MA), according to the kit's instructions. Real-time quantitative PCR was performed to detect the expression of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) using UltraSYBR (CW0957; CWBIO, Beijing, China). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The relative expression of mRNA was calculated using the 2−ΔΔCt method. The primers of the target gene were synthesized by Wuhan Hycell Biotechnology. The primer sequences are shown in Table 1.

Total RNA was extracted from PSCs using the RNAprep pure Tissue Kit (Tiangen, China) according to the manufacturer’s guidelines, and the Thermo Scientific RevertAid (Thermo Fisher Scientific, USA) was used to synthesize first-strand cDNA. The sequences of EgE2D2 and EgE2N were amplified with primers described in Table 1. Purified polymerase chain reaction (PCR) products were cloned into a pET32a (+) vector and transformed into E. coli BL21 (DE3). The BL21 strain was cultured at 37 °C, 160 rpm for 6 h, and then added with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce protein expression. Ni²⁺ affinity chromatography column (Bio-Rad, USA) was used to purify the recombinant proteins.Table 1

Primer sequences

Gene	Forward primer (5′-3′)	Reverse primer (5′-3′)
EgE2D2	CGGATCCATGGCCCTGAAGAGGATTC	CGGAATTCCTACATTGCGTACTTCTGAGTCC
EgE2N	CGAGCTCATGAGTGGACATCTTCCTACAC	CCTCGAGCTAGCGGAAGTCGGAAG
α-SMA	GACAGCTACGTGGGTGACGAA	TTTTCCATGTCGTCCCAGTTG
COL1A1	GTGCGATGACGTGATCTGTGA	CGGTGGTTTCTTGGTCGGT
COL1A2	GTTGCTGCTTGCAGTAACCTT	AGGGCCAAGTCCAACTCCTT
TIMP1	TGCAGGATGGACTCTTGCAC	GCATTCCTCACAGCCAACAG
GAPDH	CAAGGTCATCCATGACAACTTTG	GTCCACCACCCTGTTGCTGTAG

Black and bold italics signify restriction enzyme sites

Total RNA was isolated using TRIzol reagent (Vazyme, Nanjing, China). The cDNA was synthesized using a Thermo Scientific RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA) to quantify the amount of mRNA and lnc-AROD. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The cDNA was synthesized using a miRNA first-strand cDNA synthesis kit (by stem-loop; Vazyme, Nanjing, China) to quantify the amount of miRNA. Small nuclear U6 RNA was used as an internal standard. Real-time PCR analyses were performed using ChamQ Universal SYBR qPCR master mix (Vazyme, China). To determine the absolute quantity of RNA, the purified PCR product amplified from the cDNA corresponding to the lnc-AROD and IAV-M sequences was serially diluted to generate a standard curve. The primers used in this study are listed in Table S2.