Sybr green

Total RNA was extracted from cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA Cat. #12183555) following the manufacturer’s instructions and used to generate cDNA using the ReverTra Ace qPCR RT Kit (TOYOBO, Cat. #QPK-201). SYBR green (Yeasen, Shanghai, China, Cat. #11203ES03)-based qPCR was performed with the primers listed in Supplementary Table 6.

Following the producer’s directions, total RNA was isolated from cells by utilizing the RNA-Quick Purification Kit (cat# RN001, ES science). NanoDrop One (ThermoFisher Scientific) was applied to detect the concentration and purity of RNA. Subsequently, using RNA as a template, reverse transcription was conducted using PrimeScript™ RT Reagent Kit (cat# RR037A-1, Takara) to synthesize complementary DNA (cDNA). Real-time PCR was carried out with SYBR Green (cat# 11184ES08, Yeasen Biotechnology) following the manufacturer’s directions through a 7500 real-time PCR device under the following procedures: 95 °C for 2 min, 95 °C for 10 s, 60 °C for 30 s, The melting curve stage is 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 5 s, a total of 40 cycles. Each experiment was conducted three times. The relative RNA expression of USP40 and Claudin1 was calculated by 2^−ΔΔCt method. The primer sequences used for RT-qPCR were shown in Additional file 1: Table S3.

RNA was extracted using Trizol reagent and reversed transcribed into cDNA using Hifair® II 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, China). Quantitative PCR (qPCR) was performed using the reaction mix of SYBR Green (Yeasen Biotechnology) on the ABI QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA), with GAPDH serving as an internal control. Data were analysed using the 2−ΔΔCt method. The primer sequences list is reported in Supplementary Material (Table 2–3).

Total cellular RNA was extracted with Trizol (TaKaRa, Cat# 9109). Subsequently, complementary DNAs (cDNAs) were synthesized using First-Strand Synthesis System (TaKaRa, Cat# RR036A) according to the manufacturer’s instructions. cDNA was analyzed by qPCR with SYBR Green (YEASEN, Cat# 11143ES50) and gene-specific primers (mPRELID2, forward 5′-GTCGCTTGCTTCCTC-3′, reverse 5′-GATTTTCTACGCTTTCC-3′; mFUNDC2, forward 5′-GCTAACAGTCAAGGAAA-3′, reverse 5′-TCTGGAATACGAAACC-3′; mMFN1, forward 5′-ATCACTGCAATCTTCGGCCA-3′, reverse 5′-AGCAGTTGGTTGTGTGACCA-3′; mSDHA, forward 5′-GAAGATTTATCAGCGTG-3′, reverse 5′-GTGTAAGAGTGAGTGGC-3′; mKi67, forward 5′-GCTCACCTGGTCACCATCAA-3′, reverse 5′-TGACACTACAGGCAGCTGGA-3′; mAFP, forward 5′-GTTTCCAGAACCTGCCGAGA-3′, reverse 5′-CTGAGCAGCCAAGGACAGAA-3′; hHPRT, forward 5′-AGCCCTGGCGTCGTGATTA-3′, reverse 5′-ACAATGTGATGGCCTCCCA-3′; hFUNDC2, forward 5′-ACTGGCAACGAGTGGAGAAG-3′, reverse 5′-CATGCCAAGCAGAAAGCCTC-3′. Relative expression of mRNA was normalized by Succinate dehydrogenase, subunit A (Sdha) or hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA. The real-time PCR results were analyzed and expressed as relative expression of CT (threshold cycle) using the 2^−▵▵Ct method.

Total RNA extracted from HAEC with TRIzol reagent (Takara Biotechnology) was used to synthesize 1st strand cDNA with cDNA Synthesis Supermix for qPCR (Yeasen Biotech) according to the manufacturer’s instructions. SYBR Green (Yeasen Biotech) was used for real-time PCR on a Light Cycler 480 Instrument II (Roche). The relative fold change was calculated by normalizing to GAPDH using the 2^−ΔΔCt method. The primers for mRNA measurement were as follows: eNOS, forward: 5’-TTCCGCTACCAGCCAGACC-3’ and reverse: 5’-CACTCGCTTCGCCATCACC-3’; KLF2, forward: 5’-CAAGACCTACACCAAGAGTTCG-3’ and reverse: 5’- CATGTGCCGTTTCATGTGC-3’; NOX2, forward: 5’-CAGCTATGAGGTGGTGATG-3’ and reverse: 5’-GCCAGTGAGGTAGATGTTG-3’; GAPDH, forward: 5’-GGATTTGGTCGTATTGGG-3’ and reverse: 5’-GGAAGATGGTGATGGGATT-3’.

RNAs from adipose tissues were extracted using Trizol (9109, Takara, Japan). Total RNA (1 μg) was reversely transcribed with PrimeScript RT reagent Kit with gDNA Eraser kit (PR047Q, Takara, Japan) and quantitative PCR was performed using SYBR green (11143ES50, Yeasen, China) on the LightCycler480 system (Roche, Switzerland). Messenger RNA (mRNA) levels were calculated by the ΔΔCT method with the level of 36B4 or Pcdh as the internal control. The primers used for quantitative PCR are in Supplementary Table S2.

qPCR was performed according to the protocol of our laboratory^{43 (link),44 (link)}. Briefly, total mRNA was isolated from cultured cells or liver samples using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. 3ug cDNA was reverse transcribed into cDNA using Promega Kit (Promega, Madison) according to the manufacturer’s protocol. SYBR Green (YEASEN Biotech) was applied to quantify PCR amplification. Expression levels were calculated using the Δct-method. The primer pairs used in our study are described in Supplementary Table 2.