Lipofectamine rnaimax reagent kit

siRNAs for Fox and KLF16 genes used in this experiment were purchased from Sigma. The KLF 16 expression vector (pCMV-KLF16) was kindly provided by Dr. Urrutia. Brown preadipocytes and 3T3-L1 preadipocytes were transfected with siRNA or KLF 16 expression vector using the lipofectamine RNA iMAX reagent kit or lipofectamine LTX with PLUS Reagent kit (Invitrogen). The transfected cells were differentiated in differentiation medium 2 days after transfection.

To deplete JMJD2B, duplex of siRNA targeting JMJD2B (sense: 5’-CCAGUUCAGUAUCAAUUAAAGCCCG-3’, antisense: 5’-CGGGCUUUAAUUGAUACUGAACUGGAG-3’) was designed and synthesized by Integrated DNA Technologies (Coralville, Iowa, USA). The JMJD2B expression vector (pCMV-JMJD2B) was purchased from Addgene (Cambridge, MA, USA). The 3T3-L1 preadipocytes were transfected with the siRNAs or pCMV-JMJD2B using the lipofectamine RNAiMAX reagent kit or lipofectamine LTX with PLUS reagent kit (Invitrogen, Carlsbad, CA, USA). The transfected cells were differentiated in the differentiation medium 6 days after transfection.

MSCs were transfected with MIF-siRNA (sc-37137, Santa Cruz) or control siRNA (sc-37007, Santa Cruz) by means of a Lipofectamine RNAiMAX Reagent Kit (Invitrogen, 13778030) according to the manufacturer’s protocol. The transfection efficiency was determined by Western blotting 72 hours after transfection.

TLR4 siRNA (CAATTCTGTTGCTTGTATA) and control siRNA (targeting β-actin) were synthesized by Guangzhou RiboBio Co., Ltd, China. RAW264.7 cells were cultured in a 24-well plate and siRNA (50 nM) was added by Lipofectamine^® RNAiMAX Reagent Kit (Invitrogen, USA). After 24 h, the cells were incubated with Tris-HCl (pH 8.0) or Eh-rPrx (5 μg/mL).

In the knockdown experiments, gene‐specific small interfering RNA (siRNA) was designed by using the complete cDNA sequence of the porcine RNF20 gene (NCBI Gene ID: 100154259), appropriate small interfering RNA (siRNA) target sites were selected, and the corresponding primers were designed and synthesized. A nonsense codon sequence was used as a negative control. The specific siRNAs for the porcine RNF20 gene (siRNF20) and negative control (siNC) used in this experiment were purchased from Gene Pharma (Shanghai, China). Sequence information for the specific siRNAs is provided in Table S2. Porcine preadipocytes were transfected with siRNA using a Lipofectamine RNAiMAX reagent kit (Invitrogen, Carlsbad, CA, USA). The efficiency of transfection was assessed at 48 h and 72 h by Western blot.

MSCs were transfected with Sirt1-siRNA (RXO13987, TranSheepBio, Shanghai, China) or control siRNA (RXO13004, TranSheepBio, Shanghai, China) using a Lipofectamine RNAiMAX Reagent Kit (2145966, Invitrogen, California, USA) according to the manufacturer’s protocol. The lentiviral plasmid constructs for overexpression of Sirt1 in control-MSCs were purchased from TranSheepBio (Shanghai, China; Fig. S1A). The lentiviral plasmid constructs for inhibition of miR-199a-5p in IPF-MSCs were purchased from GenePharma (Shanghai, China, Fig. S1B). The lentivirus was packaged as per the manufacturer’s protocol. MSCs at a confluence of 70–80% were infected by lentivirus at a multiplicity of infection of 10 with polybrene (8 μg/ml). The transfection efficiency was determined after 72 h by Western blotting and PCR.

TGF‐β1‐siRNA (sc‐44146), Mfn2‐siRNA(sc‐43928) and control siRNA(sc‐37007) were used to transfect VPCs using a Lipofectamine RNAiMAX Reagent Kit (Invitrogen, 13778030) according to the protocol. Seventy‐two hours after transfection, VPCs were collected and the silencing efficiency was examined by Western blotting.