Pcr primers

Extraction of total RNA employed Trizol method, and synthesis of cDNA was carried out by reverse transcription. The PCR primers were generated by Shanghai Sangon Bioengineering Co., Ltd. GAPDH acted as an internal reference. LncRNA SNHG7 or EIF4G mRNA level was determined by RT-PCR (Light Cycler@480, Roche Company). The 2 ^−ΔΔCT method was applied to figure out the RNA levels [16 (link)].

Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Thermo Fisher (Beijing, China). Fetal bovine serum (FBS) and antibiotics (streptomycin and penicillin) were obtained from GIBCO (New York, NY, USA). All tissue culture plasticware was obtained from Corning (Corning, NY, USA). ISL (purity: 98.26%) was purchased from Shaanxi Green Bio-Engineering Co., Ltd. (Xi’an, China), and dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na). MTT, LPS, and oil red O were all from Sigma-Aldrich (St. Louis, MO, USA). LDL, 1 mg/mL, was obtained from Prospect (Ness Ziona, Israel) and reacted with 10 μmol/L copper sulfate for 24 h at 37 °C. After dialysis, the LDL was filtered with a 0.22 μm membrane resulting in ox-LDL; the concentration was determined using the Lowry method. Anti-CD36 and anti-ABCA1 monoclonal antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-PPARγ polyclonal antibody and anti-β-actin monoclonal antibody were from Santa Cruz Biotechnology (Delaware Ave Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson Laboratory (Main St Bar Harbor, ME, USA). The enhanced chemiluminescence (ECL) kit was purchased from GE Healthcare (Waukesha, WI, USA). PCR primers were from Sangon Biotech (Shanghai, China). All other chemicals were of the best grade and obtained from commercial sources.

Total RNA of HK-2 cells was extracted using Trizol reagents (Invitrogen, Carlsbad, CA). Extracted RNA was reverse-transcribed to complementary DNA (PrimeScript; TaKaRa, Kyoto, Japan), which was utilized for real-time polymerase chain reaction (PCR) (SYBR reagents, TaqTMTaKaRa). PCR primers (Sangon, Shanghai, China) were designed with the following sequences: HIF-1α forward: 5′-CGGAGGTGTTCTATGAGCTGG-3′, reverse: 5′-AGCTTGTGTGTTCGCAGGAA-3′. HIF-2α forward: 5′-CGGAGGTGTTCTATGAGCTGG-3′, reverse: 5′-AGCTTGTGTGTTCGCAGGAA-3′. PHD2 forward: 5′-AGGCGATAAGATCACCTGGAT-3′, reverse: 5′-TTCGTCCGGCCATTGATTTTG-3′; 18s forward: 5′-CGGCTACCACATCCAGAA-3′, reverse: 5′-CCTGTATTGTTATTTTTCGTCACTACCT-3′. 18s mRNA was tested as endogenous control for desired mRNAs. The relative gene expressions were calculated in accordance with the ^ΔΔCt method [33 (link)]. Relative mRNA levels were expressed as 2^−ΔΔCt and ratios to internal control.

Total RNA was extracted with TRIzol reagent. Total RNA concentrations were measured using ND-1000 (NanoDrop Technologies), and cDNA was synthesized with high-capacity cDNA reverse transcription kits (Applied Biosystems, F. Hoffmann-La Roche Ltd., Switzerland), according to the protocol provided by the manufacturer. PCR primers for TGF-β1, Smad2, Smad3, Smad7, and GAPDH were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the forward and reverse primers used for amplification are shown in Table 1. Real-time PCR was performed using a one-step real-time PCR system (Applied Biosystems) with SYBR green fluorophore. Each sample for reactions was run in at least duplicates. Threshold cycle (Ct) data were collected by an inherent system and posted on an Excel sheet. GAPDH was performed as an internal control. 2^−ΔΔCt was calculated to evaluate mRNA fold change relative to GAPDH [26 (link)].