Dmem f12 complete medium

HK2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in 10% foetal bovine serum (Gibco, USA)-containing Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) complete medium (Corning, USA). When the cells reached 50% confluence, they were synchronised overnight in serum-free DMEM/F12 medium, which was then replaced with complete medium containing 8 ng/ml recombinant human TGF-β1 (PeproTech Inc., USA), before the cells were cultured for 48 h and 72 h to induce fibrosis. miR-294, miR-133 and control miRNA (GeneCopoeia Inc., Rockville, MD, USA) were transfected into synchronised HK2 cells in the miRNA transfection groups with the jet-PRIME® transfection reagent (Polyplus Transfection, Inc., USA) according to the manufacturer’s instructions. After 24 h of transfection, the medium was replaced with complete medium containing 8 ng/ml recombinant human TGF-β1, and the cells were incubated for an additional 48 h and 72 h.

Primary head kidney leukocytes were isolated by using a discontinuous Percoll gradient as previously described (37 (link)). The leukocytes cultured in a six-well plate (Corning, United States) with DMEM-F12 complete medium [DMEM-F12 with 10% fetal bovine serum and 1% Pen/Strep (penicillin/streptomycin)] in a CO₂ incubator at 28°C. The leukocytes (1 × 10⁷/well) were treated with LPS (100 µg/mL), poly(I:C) (50 µg/mL), and ATP (100 µM or 1 mM), respectively. Each condition was done in quadruplicate. Cell samples were collected at 6, 12, 24, and 48 h after stimulation, and total RNA was then reversed to cDNA for qPCR. ATP was purchased from Sigma–Aldrich, and all cell culture reagents were purchased from Gibco (USA).