Igf 2

Total proteins from the blood samples of AS patients and HVSMCs were isolated using RIPA lysis buffer containing a protease inhibitor (Sbjbio^® life science). The concentrations of extracted proteins were determined with a BCA protein assay kit (Sbjbio^® life science). Then, polyacrylamide gels were used to separate protein with a SureLock Midi-Cell Electrophoresis System. After blocking the aspecific signals using skimmed milk (Yili, Beijing, China), polyvinylidene fluoride membranes with protein bands were incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA) (#13-3900; 1:5000; Thermo Fisher, Waltham, MA, USA), matrix metalloprotein 2 (MMP2) (#436000; 1:250; Thermo Fisher), IGF2 (#MA5-17096; 1:1,000; Thermo Fisher), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#MA1-16757; 1:2,000; Thermo Fisher). After being exposed to eyoECL Plus (Beyotime, Shanghai, China), the visualized blots were analyzed using Image J software.

IL-2 (Miltenyi Biotec); IL-21, IL-6, IFN-α, TGF-β3, SDF1α, Osteoprotegerin, M-CSF, Osteopontin, SCF, MIF, IGF-BP2, IGF-II, and Activin A (PeproTech); Jagged-1, ANGPT1, VEGF-165 and VEGF-121 (R&D Systems); Multimeric-APRIL (Caltag); goat anti-human IgM & IgG F(ab′)₂ fragments (Jackson ImmunoResearch); Hybridomax hybridoma growth supplement (Gentaur); Lipid Mixture 1, chemically defined (200×) and MEM Amino Acids Solution (50×) (Sigma-Aldrich); SB525334 (Selleckchem).

Schwann cells from P1 STAT3 cKO and WT mice were assayed as in Meier et al. (1999 (link)). Cells were plated at low density (200 cells/coverslip) or high density (3000 cells/coverslip. After 3 h at 37°C and 5% CO₂, one set of coverslips from each animal was fixed immediately for immunolabeling to obtain a reference point for the quantification of survival at later time points. The remaining sets were topped up with 400 μl of defined simple medium (sDM) (Meier et al., 1999 (link)) alone or sDM containing 1.6 ng/ml IGF-II (Peprotech), 0.8 ng/ml PDGF-BB (Peprotech), and 0.8 ng/ml NT-3 (Regeneron Pharmaceuticals), or conditioned medium, and cultured for 48 or 72 h. Then, cells were fixed using 4% PFA for 10 min, labeled with S100 antibodies and Hoechst dye, and the number of surviving Schwann cells counted. Survival percentage is the number of living cells present at 48 and 72 h as a percentage of the number of cells that had attached to the substrate in sister cultures at 3 h. sDM consists of 1:1 DMEM and Ham's F-12 supplemented with BSA (350 μg/ml). Schwann cell conditioned medium was prepared as previously described (Meier et al., 1999 (link)).

To study whether ME-CSCs can differentiate into keratinocyte-like cells, they were plated at a density of 3 × 10³ cells/cm² in a 12-well and cultivated in DMEM low glucose (Sigma-Aldrich) containing 10% FCS, keratinocyte growth factor (KGF, 10 ng/ml; Peprotech) and epidermal growth factor (EGF, 20 ng/ml; Peprotech). After 3 days of culture, hepatocytes growth factor (HGF, 10 ng/ml; Peprotech) and insulin-like growth factor-2 (IGF-II, 60 ng/ml; Peprotech) were added. ACSCs cells served as control. After 14 days, RNA isolation and immunocytochemical stainings were performed as described above.

RLSCs [17 (link)] were grown in DMEM (Dulbecco's Modified Eagle's Medium) (GIBCO Life Technology, Monza, Italy) with 10% FBS (Sigma Aldrich, St. Louis, MO), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), and antibiotics on collagen I (GIBCO Life Technology, Monza, Italy) coated dishes (BD Falcon, Franklin LAkes, NJ, USA) and on 0.4 kPa and 80 kPa hydrogels obtained as described above. Nontumorigenic murine MMH/E14 and WT/3A hepatocytes [19 (link), 20 (link)] were grown in RPMI 1640 with 10% FBS (GIBCO Life Technology, Monza, Italy), 50 ng/mL EGF, 30 ng/mL IGF II (PeproTech Inc., Rocky Hill, NJ, USA), 10 μg/mL insulin (Roche, Mannheim, Germany), and antibiotics, on collagen I (GIBCO Life Technology, Monza, Italy) coated dishes (BD Falcon, Franklin Lakes, NJ, USA) and on 0.4 kPa and 80 kPa hydrogels obtained as described above. 200.000 cells were seeded on each 25 mm coverslip with hydrogel. The morphological analysis was performed by phase-contrast microscopy.