Dmem medium

HuH-7 cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin. HepG2-NTCP cells were generated by NTCP expressing lentivirus and maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 ug/ml streptomycin and 20ug/ml Blasticidin (Invivogen). Cells were transfected with plasmids by PolyJet™ In Vitro DNA Transfection Reagent (Signagen) according to the Manufacturer's instructions.

The normal human osteoblast (hFOB) and OS cell lines (MG63, U2OS, HOS, Saos-2) were obtained from the ATCC (Manassas, USA). All the OS cells were maintained in the DMEM Medium (Invitrogen; USA) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin. While hFOB cells were cultured in DMEM Medium/Nutrient Mixture F-12 (DMEM/F-12) containing 10% FBS and 0.3 mg/ml G418 (Invitrogen, USA). All the cells were stored in the humidified atmosphere with 5%CO₂ at 37°C.

Human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany) and grown in endothelial cell growth medium (PromoCell) supplemented with gentamycin (50 µg mL⁻¹) and amphotericin B (50 ng mL⁻¹). For all experiments, we used HUVECs pooled from up to four donors, which did not exceed passage 4. Human embryonic kidney (HEK) 293 cells (ATCC CRL-1573) were grown in DMEM medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS), 100 IU mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin (Invitrogen, Cergy Pontoise, France). C166 mouse endothelial cells (accession number CRL-2581) and LLC1 mouse lung cancer cells (accession number CRL-1642) were grown in DMEM medium (Invitrogen, Cergy Pontoise, France). Media were supplemented with 10% fetal calf serum (FCS), 100 IU mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin. As positive control for apoptosis assays, LLC1 mouse lung cancer cells were treated with 100 nmol/L Staurosporine (Sigma, St. Louis, MO, USA) overnight. For RNA isolation and quantitative RT-PCR experiments, HUVEC and LLC1 cells were maintained for 48 h (HUVEC) or 24 h (LLC1) in medium in the presence of GW0742 (Selleckchem, Houston, TX, USA) or GSK3787 (Selleckchem) dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 µmol/L. Controls were treated with vehicle (0.1% DMSO) only [6 (link),16 (link)].

Primary MuSCs were isolated as previously described.²⁴ Briefly, TA muscles from 3‐month‐old mice were dissected and dissociated with collagenase (Roche, Indianapolis, IN, USA). The cells were negatively selected by biotinylated CD45, CD11, CD31 and Sca1 antibodies. The muscle cells in the flowthrough were subjected to CD34‐FITC (BD biosciences) and integrin α₇‐allophycocyanin (R&D systems, Minneapolis, MN, USA) staining. The viable PI⁻CD34⁺integrin‐α₇⁺ MuSCs were collected by FACS sorting (Influx, BD biosciences, Franklin Lake, NJ, USA). MuSCs were cultured on collagen coated dishes in F10 medium containing 10% foetal bovine serum (FBS), 5 ng/mL IL‐1α, 5 ng/mL IL‐13, 10 ng/mL IFN‐γ, and 10 ng/mL TNF‐α (R&D Systems), and 2.5 ng/mL FGF (Invitrogen, Red Wood City, CA, USA) as described previously.²⁴ MuSCs were differentiated in differentiation medium [DMEM medium (invitrogen)] containing 2% horse serum (HyClone, Malborough, MA, USA) for 3 days. C2C12 cells (ATCC) were cultured in DMEM medium (Invitrogen) containing 10% FBS (HyClone) and differentiated in DMEM medium (Invitrogen) containing 2% horse serum (HyClone) for 3 days. The differentiated myotubes were further isolated by pre‐attaching to plates for three times.

hfC-MSCs were treated with adipogenic medium consisting of DMEM medium (Gibco-Invitrogen) containing 10% FBS (Hyclone), 500mM Isobutylmethylxanthine, 1mM dexamethasone, 10mg/ml insulin and 100mM Indomethacin (Adipogenesis kit, Chemicon). The control cells were treated with DMEM medium (Gibco-Invitrogen) containing 10% FBS (Hyclone) alone. After 18 days the experimental and control cells were fixed and stained with Oil-Red O Stain to visualize fat droplets in the cells.

Bone marrow (BM) cells were flushed from femur and tibia of Adrb2^+/+ and Adrb2^−/− mice and cultured using DMEM supplemented with 20% (v/v) FBS, 1% (v/v) P/S, 20 ng/ml mouse recombined murine M-CSF (Proprotech, USA) for 7 days (17 (link)). The purity of BM-macrophage cultures was confirmed by FACS using CD11b (1:100, BioLegend, San Diego, CA, USA) and F4/80 (1:100, BioLegend, San Diego, CA, USA) antibodies. Seven days later, BM-derived macrophages were cultured in a DMEM medium (Invitrogen, Carlsbad, CA, USA) for 24 h to remove the residual M-CSF. M0 macrophages (non-differentiated macrophages) were then stimulated with recombinant IL-4 (rIL4) (20 ng/ml, Proprotech, USA) with or without mTORC1 inhibitor rapamycin (100 ng/ml, MCE, USA) for 24 h. After that, the rIL-4 was removed and the cells were washed to remove the remaining rIL-4 and collected for further analysis. For measuring IL-4, the cells were washed and the DMEM medium (Invitrogen, Carlsbad, CA, USA) was added for another 24 h.

The colon cancer cell line HT29, carrying mutant BRAFV600E and PIK3CAP449T, was obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 1% glutamine (Invitrogen, Milan, Italy). The patient-derived colon cancer cell lines: Pt-1 carrying mutant BRAFV600E and PIK3CAP449T; Pt-2 carrying wild type (wt) BRAF and mutant PIK3CAH1047R; Pt-3 carrying mutant KRAS G13 and mutant PIK3CAE542K were kindly provided by Istituto Superiore di Sanità (ISS) and maintained in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 1% glutamine (Invitrogen, Milan, Italy). All the cell lines, with the exception of Pt-3 cells, carry mutant p53.