Primescript rt mix

Total RNA from HepG2 cells was isolated using RNAiso Plus (Takara). cDNA was synthesized using the PrimeScript RT Mix (Takara) kit according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the NovoStart SYBR qPCR SuperMix Plus kit (novoprotein, Suzhou, China). NEAT1 gene expression was normalized to GAPDH and relative quantification was determined using the 2^−∆∆Ct method. The sequences of primers are listed in Table S1.

Total RNA was extracted from HCAECs using the RNeasy mini kit (Qiagen, Germany). cDNA was synthesized using the PrimeScript RT Mix (Takara, Japan). The mRNA expression levels of plexin C1, integrin β1, TNF-α, IL-1β, IL-6, and IL-18 were analyzed using SYBR Green Realtime PCR Mix (Takara, Japan) on an ABI 7500 analyzer (ABI, USA).

Total RNA from HCAECs was extracted by using the RNeasy Mini Kit (Qiagen, Germany) and then reverse-transcribed to complementary DNA by using the PrimeScript RT Mix (Takara, Dalian, China). The mRNA expression levels of plexin B1, plexin B2, IL-1β, IL-6, and IL-8 were analyzed using the SYBR Green Real-time PCR Mix (Takara, China) in the ABI 7500 analyzer (ABI, CA, USA).

Total RNA was reversely transcribed using a PrimeScript RT Mix (Takara, China) in accordance with the manufacturer’s instructions. Following RT-PCR amplification, melting curve analysis was executed to identify the specific generation of the PCR product with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as internal controls. Fold changes for aberrant expression were calculated using the 2–ΔΔCt method. Reactions were performed in 20 μL system including SYBR Select Master Mix(Thermo Fisher Scientific) and 40 ng cDNA with the following reaction conditions: 95 °C 10 minute, 40 cyclesof 95 °C 30 sec, 58 °C 1 minute, 72 °C 1 minute. The primer used for the amplification of the human AK2 gene was as follows: forward primer, 5′-GCAGAACCCGAGTATCCTAAAGG, and reverse primer, 5′-TTCCCAGCATCCATAGTTGCC. GAPDHwas used as the housekeeping gene with forward primer 5′-TTCGACAGTCAGCCGCATCTTCTT and reverseprimer 5′-ACCAAATCCGTTGACTCCGACCTT. Delta-delta Ct values were used to determine their relative expression as fold changes, as previously described^{24 (link)}.

Total RNA was isolated from cells using Easy-Blue™ total RNA isolation kit (iNtRON Biotech) following the manufacturer’s instructions. 5 × PrimeScript™ RT mix (TaKaRa) was used during reverse transcription to acquire cDNA. Quantitative real-time PCR was performed using 2 × TB-Green premix (TaKaRa) by LightCycler-480^®II (Roche). Rn18s gene was used as internal loading control for normalizing gene expression data (Table 1).Table 1

Sequences of primers for quantitative real-time PCR

Primer pairs		Primer sequences (5’-3’)
Ptpn11	Forward	AGTCCAAAGTGACCCACGTC
	Reverse	CCATCATGCAGAACGACCCT
Dppa3	Forward	CGTACCTGTGGAGAACAAGAGTG
	Reverse	CA TTCTCAGAGGGA TCCCA TCTTTG
Rex1	Forward	CTTCGAAAGCTTGGAGGAAGTGGAG
	Reverse	GGACACTCCAGCATCGATAAGACAC
Nr0b1	Forward	ACAGAGCAGCCACAGA TGGTGTC
	Reverse	GATGTGCTCAGTAAGGATCTGCTG
Klf4	Forward	GAACAGCCACCCACACTTGTGAC
	Reverse	CTGTCACACTTCTGGCACTGAAAG
Esrrb	Forward	GATTCTCATCTTGGGCATCGTGTAC
	Reverse	CTGACTCAGCTCATAGTCCTGCAG
Klf2	Forward	CACACATACTTGCAGCTACACCAAC
	Reverse	CAAGTGGCACTGAAAGGGTCTGTG
Fgf5	Forward	CATCGGTTTCCATCTGCAGATCTAC
	Reverse	GTTCTGTGGATCGCGGACGCATAG
Cer1	Forward	GTGGAAAGCGA TCA TGTCTCA TCG
	Reverse	GCAAAGGTTGTTCTGGACAACGAC
Pou5f1	Forward	GAGAAAGCGAACTAGCATTGAGAAC
	Reverse	TGTAGCCTCATACTCTTCTCGTTG
Sox2	Forward	ATGGGCTCTGTGGTCAAGTC
	Reverse	CCCTCCCAATTCCCTTGTAT
Nanog	Forward	GTGCACTCAAGGACAGGTTTCAG
	Reverse	CTGCAATGGATGCTGGGATACTC
c-Myc	Forward	ACCACCAGCAGCGACTCTGA
	Reverse	TGCCTCTTCTCCACAGACACC

Total RNA was extracted from NP cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA templates by PrimeScript™ RT Mix (RR036A, TaKaRa, Tokyo, Japan). RT-PCR assay was performed to assay relative gene expression containing superoxide dismutase1 (SOD1), SOD2, catalase (CAT), glutathione peroxidase1 (GPX1), MMP3, MMP13, ADAMTS4, and ADAMTS5 by normalization of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) according to 2^−ΔΔCt methods. The primers of the genes were listed in Table 1.

Total RNA was isolated using the TaKaRa Mini BEST Universal RNA Extraction Kit (Takara, Dalian, China). A NanoDropND 2000 spectrophotometer was used to quantify total RNA concentrations (Thermo Scientific, Wilmington, DE, USA). Total RNA (1 µg) was then reverse-transcribed using PrimeScript RT Mix (Takara, Dalian, China). A C1000 Touch thermal cycler (BIO-RAD) was used to perform RT-qPCR analysis using TB Green Premix Ex TaqII (Thermal Cycler DiceTMReal Time System, Otsu, Japan). The PCR mix had a final volume of 25 μL and included pre-denaturation at 95 °C for 30 s, denaturing at 95 °C for 5 s, and annealing at 60 °C for 30 s as the thermal cycling conditions. The primer sequences for each target gene are listed in Table 1. The amplification efficiency of each gene was optimized to maximize reactions.