Dmem f12

A549 cells and MCF10A cells were obtained from cell bank of Chinese Academy of Sciences and have been test to be free of mycoplasma contamination. The identities of cells have been confirmed by STR profiling. A549 cells were maintained in Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-12 (DMEM/F-12) (Corning) supplemented with 10% fetal bovine serum (FBS). MCF-10A cells were maintained in Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-12 (DMEM/F-12) (Corning) supplemented with 5% Horse Serum (Invitrogen #16050-122), 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 100 ng/ml Cholera Toxin, 10 μg/ml Insulin, and 1% Pen/Strep (100× solution, Invitrogen #15070-063). Importantly, numerous studies have shown that there is substantial amount of TGF-β1 in FBS, which is sufficient to induce partial EMT^{26 (link),41 (link)}. Moreover, MCF10A cells could undergo spontaneous EMT when cultured at low confluence^{42 (link),43 (link)}. To successfully induce EMT, cells were seeded in serum-free medium for 24 h to reach ∼70% confluence before subject to TGF-β1 treatment. TGF-β1 (Gibco^® PHG9214) was added to a final concentration of 4 ng/ml and medium with TGF-β1 was replaced every other day.

Human proximal tubular cells (HK2 cells) were obtained from ATCC and grown in DMEM/F12 (Corning) containing 10% FBS (Quacell Biotechnology) and 1% penicillin/streptomycin (Corning) and maintained at 37°C in 5% CO₂ atmosphere. Mouse kidney proximal tubular cells (mPTCs) were prepared as previously described (57 (link)). Briefly, mPTCs were isolated from 10-week-old C57BL/6J mice and cultured in DMEM/F12 (Corning) containing 10% FBS (Quacell Biotechnology) at 37°C in 5% CO₂ atmosphere.
The HK2 cells and mPTCs were seeded on 6-well plates (Thermo Fisher Scientific) at a density of 2 × 10⁵ cells/well for 24 hours and were serum-starved for 12 hours. The medium was changed to fresh serum-free DMEM/F12 before treatment. For morphological assessments, the cells were fixed with 4% paraformaldehyde and photographed using a microscope (Leica) connected to a digital camera with a macroconversion lens. For profibrotic response experiments, HK2 cells and mPTCs were treated with cyclic stretch for 24 hours or TGF-β₁ (5 ng/mL, R&D Systems) for 48 hours, accompanied with either GsMTx4 (1 μM or 5 μM, TAIJIA biotech) or Piezo1 siRNA (RIBOBIO) pretreatments. Yoda1 (0.2 μM, MedChemExpress) was used to stimulate HK2 cells for 1 to 24 hours, with or without Piezo1 siRNA. A total of 1 μM Yoda1 was used to stimulate mPTCs for 24 hours. All the cells were then collected for the protein or RNA analyses.

Primary hepatocytes isolated from C57BL/6 or Mir-122 whole-body knockout mice (Mir-122 KO) using a two-step collagenase perfusion (46 (link)) were seeded at 1.5 × 10⁵ viable cells per well in 24-well Primaria Cell Culture plates (Corning) in seeding media: DMEM-F12 with 15 mM HEPES (Corning), 5% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), and Antibiotic-Antimycotic Solution (Corning). After 4 hrs, seeding media were replaced with maintenance media: DMEM-F12 with 15 mM HEPES (Corning), 0.5% FBS (Atlanta Biologicals), Antibiotic-Antimycotic Solution (Corning), and ITS Supplement (containing 1 µg/mL insulin, 0.55 µg/mL transferrin, and 0.67 ng/mL sodium selenite; Roche). Vero E6 (in DMEM-10) and HepG2 (in MEM-10) cells were seeded on 24-well plates at 1 × 10⁵ cells per well. After 16–24 h of plating, cells were infected with either RVFVmiR-184 or RVFVmiR-122 (MOI = 0.1) for 1 h at 37°C, washed once with PBS, and incubated with fresh medium. Supernatants were collected at various times post-infection and used for FFU assay as described above.

Minced mammary tissue was digested (1.5 mg/ml collagenase A (Roche, San Francisco, CA; 75 μg/ml DNase I, Roche; 1 mg/ml hyaluronidase, MP Biomedicals, Santa Ana, CA) in growth media (10 % fetal bovine serum [FBS], DMEM/F-12, penicillin G/streptomycin sulfate/amphotericin B) at 37 °C for 3 h. Organoids (40–100 μm diameter) were plated in primary porcine mammary epithelial media (modified from MEGM [21 ] as a 1:1 mix of MCDB170 (US Biological, Salem, MA) and DMEM/F-12 (CellGro, Manassas, VA) with penicillin G/streptomycin sulfate/amphotericin B, 0.5 % FBS, bovine insulin (7.5 μg/mL, Sigma-Aldrich), human EGF (5 ng/mL, Millipore, Billerica, MA), hydrocortisone (0.25 μg/mL, Sigma-Aldrich), human apo-transferrin (2.5 μg/mL, Sigma-Aldrich), ethanolamine (0.1 mM, Sigma-Aldrich), o-phosphoethanolamine (0.1 mM Sigma-Aldrich), bovine pituitary extract (35 μg/mL, Gemini Bio-Products, West Sacramento, CA), and lipid-rich bovine serum albumin (0.1 %, Gemini Bio-Products).