Lipofectamine 6000 reagent

pGL3-CDH1, pGL3-CD44, pGL3-TP53, pGL3-CAV1, and phRL-SV40 were co-transfected into overexpressed or knocked down SRA1 isoform HepG2 cells by using Lipofectamine 6000 reagent (Beyotime, China) according to the manufacturer’s protocol. 48 h after transfection, the cells were lysed, and luciferase activity was measured using a Dual-Luciferase Reporter Gene Assay Kit (Beyotime, China).

The human lung cancer cell lines A549 and H1299 were purchased from the American Type Culture Collection (ATCC). All cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fischer Scientific, Inc.) at 37 °C in the presence of 5% CO₂.
Lung cancer cells were seeded in 6-well plates and grown for 24 h. Then, the cells were transfected with 2.5 μg of shRNA using Lipofectamine 6000 reagent (Beyotime, Shanghai, China) following the manufacturer’s protocol.

Primary hepatocytes were isolated from 8 week-year-old C57BL/6 mice using a two-step collagenase perfusion method as previously described [27 (link)]. Briefly, mice were sacrificed with the livers harvested and perfused with collagenase buffer. Next, the livers were gently shacked to release the hepatocytes into the medium. Then, the cells were washed and purified with a 50% Percoll solution (#17-0891-01; GE Healthcare Life Sciences, Pittsburgh, PA, USA). To mimic NAFLD stimulation in vitro, primary hepatocytes were stimulated with 0.5 mmol/L palmitic acid plus 1.0 mmol/L oleic acid (PO) for 24 h [27 (link)]. To verify the role of miR-137-3p, primary hepatocytes were preincubated with miR-137-3p antagomir (50 nmol/L) for 24 h using the Lipofectamine 6000™ reagent (#C0526; Beyotime) before PO stimulation. For PDE4D silence, cells were pretransfected with the small interfering RNA against PDE4D (siPDE4D) or siRNA for 24 h before miR-137-3p antagomir treatment [40 (link)–42 (link)]. The siPDE4D (#sc-152130) and matched siRNA were purchased from Santa Cruz Biotechnology (Danvers, MA, USA).

miRNAs (miR-150-5p mimics, miR-150-3p mimics, and negative control) were obtained from RiboBio (Guangzhou, China) and referred to as miR-150-5p, miR-150-3p and miR-NC, respectively. siRNAs were obtained from GenePharma (Shanghai, China). The sequences of siRNAs are listed in Supplementary Table S1. miRNAs and siRNAs were transfected at a final concentration of 50 nM and 30 nM respectively, using Lipofectamine 6000 Reagent (Beyotime). The cells were then collected 48 h after transfection for subsequent analysis.

The wild type or mutant 3′-UTR of Cab39 was cloned into the pGL3 plasmid (Promega; Madison, WI, USA) containing a luciferase report gene, which was then cotransfected with the miR-31-5p agomir or AgNC using the Lipofectamine 6000 Reagent (Beyotime; Shanghai, China). The cells were cultured for 48 h and then collected to detect the firefly and Renilla luciferase activities by a Dual-Luciferase Reporter Assay System (Promega; Madison, WI, USA) [31 (link)–33 (link)].

MH-S, a murine alveolar macrophage cell line, was purchased from ATCC (Manassas, VA) and cultured in RPMI 1640 medium containing 10% fetal bovine serum. Cells were preincubated with miR-31-5p agomir (50 nmol/L), antagomir (100 nmol/L), or their negative controls for 24 h using the Lipofectamine 6000 Reagent (Beyotime; Shanghai, China), followed by LPS stimulation (100 ng/mL) for an additional 6 h as previously described [14 (link)]. For AMPKα inhibition, the cells were pretreated with CpC (20 μmol/L) at 12 h before miR-31-5p manipulation [20 (link)]. For Cab39 silence, the macrophages were preinfected with lentivirus carrying shCab39 (MOI = 50) or shCtrl for 4 h and then maintained for an additional 24 h before miR-31-5p manipulation.