Ham s f10

Mouse myoblast cultures were prepared from skeletal muscle removed from the hind limbs of three- to four-week-old C57BL/6 mice, as previously described [41 (link)]. The muscle tissue was finely minced with scissors in a digestion buffer (Ham’s F-10 (Gibco), 400 U/mL penicillin, 400 µg/mL streptomycin, 1 µg/mL amphotericin B (Gibco), and 2.5 mM calcium chloride). An amount of 10 mg/mL Collagenase D (Roche) and 2.4 U/mL Dispase II (Roche) were added, and the tissue incubated for two hours at 37 °C. The muscle slurry was then pre-plated using plain tissue-culture flasks: twice for 20 min, once for 40 min, then at 24 h intervals for the next five days in myoblast growth media (Ham’s F-10 (Gibco), 20% fetal bovine serum (Gibco), 2.5 ng/mL recombinant human basic fibroblast growth factor (bFGF), 2 mM L-glutamine (Gibco), 100 U/mL penicillin, and 100 μg/mL of streptomycin (Gibco)). Myoblasts were maintained in growth media at 37 °C under 5% CO₂ and passaged at 80% confluence with a dissociation buffer (8.5 mM NaCl, 0.5 mM KCl, 2.3 mM NaHCO₃, 0.8 mM NaH₂PO₄.2H₂O, 0.56 mM glucose, 0.096 mM EDTA, 10 ng/mL phenol red, and trypsin (Life Technologies) at 0.25%) and resuspended in myoblast proliferation media.

Adult human myoblasts were from a young adult [14 (link)], cultured from passage 10 to 14 in Ham's F-10 (Gibco), 10% Bovine Growth Serum (Hyclone), 30 ng/mL FGF2, and 1% penicillin–streptomycin on Matrigel (BD Biosciences) coated plates (1:100 Matrigel:PBS), at 37C and 5% CO2. For experimental conditions involving immunostaining, human cells were plated at 10,000 cells/well in Matrigel coated 8-well chamber slides (1:100 Matrigel: PBS), and cultured for 72 hours with daily re-feedings at 37C in 10% CO2 incubator prior to fixation with 70% ethanol at 4°C. Mouse myoblasts were cultured and expanded in mouse growth medium: Ham's F-10 (Gibco), 20% Bovine Growth Serum (Hyclone), 5 ng/mL FGF2 and 1% penicillin–streptomycin on Matrigel coated plates (1:300 Matrigel: PBS), at 37°C and 5% CO2. For experimental conditions involving immunostaining, mouse cells were plated at 40,000 cells/well on Matrigel coated 8-well chamber slides (1:100 matrigel: PBS) and cultured for 24 hours at 37°C in 10% CO2 incubator prior to fixation with 70% ethanol at 4°C. All experiments using a MEK inhibitor were treated with 10 μM MEK1/2 Inhibitor (U0126, Cell Signaling Technologies).
Human embryonic stem cells (H9 and H7 lines), were cultured in mTeSR-1 (Stem Cell Technologies) as published [17 (link)], and the heparin binding fraction of hESC-Conditioned Medium was prepared as published [17 (link)].

Hepatic stem-like epithelial cells (LE/6) were a generous gift from Prof. Nelson Fausto. They were cultured in Dulbecco’s minimal essential medium: Ham’s F-10 (1:1) (Invitrogen, Waltham, MA) supplemented with 1 μg/ml insulin (Sigma, St Louis, MO) and 0.5 μg/ml hydrocortisone (Sigma). 293 T and Huh7 cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China), and maintained in DMEM (Gibco, USA) medium. All cell cultures were supplemented with 10% fetal bovine serum (Gibco), 100 IU/ml penicillin and streptomycin in 5% CO₂ at 37 °C. Lentivirus was packaged in 293 T cells. 48 h after the cotransfection, virus-containing supernatants were collected and incubated with LE/6 cells in the presence of 10 μg/ml polybrene for 24 h. HBx-stably overexpressing (LE/6-HBx) and control (LE/6-vec) cells were achieved by selection with 5 μg/ml puromycin for 2 weeks. LE/6-vec and LE/6-HBx cells were then treated with 5 ng/ml or 10 ng/ml TGF-β1 for 8 weeks, and renamed LE/6-vec + T and LE/6-HBx + T cells, respectively. Cells stably with miR-199a-3p overexpression or knockdown and the control vectors were generated by selection with 500 μg/ml hygromycin or neomycin for 2 weeks.

MRC5 human fibroblasts were cultured in a 1:1 ratio of Dulbecco’s modified Eagle medium and Ham’s F10 (Invitrogen) supplemented with 10% foetal calf serum (Biowest) and 1% penicillin–streptomycin (Sigma-Aldrich) at 37 °C and 5% CO₂ in a humidified incubator.
TIG3 cells were grown in Dulbecco’s modified Eagle medium containing 10% FBS and 1% penicillin–streptomycin supplemented with MEM non-essential amino acid mix. Quiescent cells were obtained by contact inhibition. SA-β-galactosidase assay was performed using Senescence β-Galactosidase Staining Kit from Cell Signaling, following manufacturer instructions.
mESCs were maintained in 2i medium deficient in lysine, arginine and l-glutamine (PAA) at 37 °C and 5% CO₂ in a humidified incubator. For SILAC labelling, cells were grown in a medium containing 73 µg ml⁻¹ light [¹²C₆]-lysine and 42 µg ml⁻¹ [¹²C₆, ¹⁴N₄]-arginine (Sigma-Aldrich) or similar concentrations of heavy [¹³C₆]-lysine and [¹³C₆, ¹⁵N₄]-arginine (Cambridge Isotope Laboratories).