Transforming growth factor β1

Human umbilical vein endothelial cells (HUVECs) (BCRC H‐UV001) at passages 3–6 were cultured in M199 medium (Gibco) that contained 20% Fetal Bovine Serum (Hyclone), 1% ECGS (Millipore), 0.2% Heparin (China Chemical & Pharmaceutical Co., Ltd), 100 μg/ml streptomycin and 100 units/ml penicillin (Gibco). In detail, 1 × 10⁶ HUVEC cells were seeded in a 10‐cm dish. After overnight incubation, cells were treated with 10 ng/ml of transforming growth factor (TGF)‐β1 (R&D), 2 μM of monocrotaline (MCT) (Sigma) or 20 ng/ml propylthiouracil (PTU) (Panbiotic Laboratories Co., Ltd.). After additional incubation for 48 h, the cells were collected, and protein extract was harvested with RIPA buffer for Western blot analysis.

Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R&D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). A mouse monoclonal antibody (clone 3C9) that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, San Francisco, CA). RPMI 1640 tissue culture medium, foetal calf serum (FCS) and L-glutamine were purchased from SAFC Biosciences (Lenexa, KS).

pMSCs and eMSCs were induced to differentiate into osteogenic, chondrogenic, and adipogenic lineages. For osteogenic differentiation, both types of cells were cultured in osteogenic medium (DMEM [Dulbecco's modified Eagle's medium] supplemented with 10% fetal bovine serum (FBS), 50 μM ascorbic acid‐2‐phosphate, 10 mM β‐glycerophosphate, and 50 nM dexamethasone [all from Sigma‐Aldrich]). The medium was changed every 3 days. After 3 weeks, the cells were fixed in 4% paraformaldehyde and processed for Alizarin Red S and Von Kossa staining. For chondrogenic differentiation, cells were cultured in chondrogenic medium (DMEM supplemented with 10% FBS, 50 μM ascorbic acid‐2‐phosphate [Sigma‐Aldrich], and 10 ng/ml transforming growth factor [TGF]‐β1 [R&D Systems, Minneapolis, MN,
https://www.rndsystems.com]). Cells were processed for Safranin O and immunocytochemistry staining for COL2A1 and Aggrecan after 3 weeks. For adipogenic differentiation, cells were exposed to adipogenic medium (DMEM supplemented with 10% FBS, 1 μM dexamethasone, 200 μM indomethacin [Sigma‐Aldrich], 10 μg/ml insulin [Sigma‐Aldrich], and 0.5 mM methylisobutylxanthine [Sigma‐Aldrich]) for 4 weeks. The medium was changed every 3 days. After 4 weeks, the cells were processed for Oil Red O and immunocytochemistry staining of aP2 and C/EBP.

The clear cell renal carcinoma (Caki-2) cell line derived from the epithelium of the proximal tubules, the in vitro spontaneously transformed aneuploidy immortal keratinocyte (HaCaT) cell line from adult human skin, the spontaneously arising retinal pigment epithelial (ARPE-19) cell line, the colon carcinoma (Caco-2) cell line and the normal human small intestinal epithelial cell-6 (HIEC-6) were obtained from the American Type Culture Collection (Manassas, VA, USA. RPMI 1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA) and fetal bovine serum (FBS) from Bio Whittaker (Walkersville, MD, USA). L-glutamine, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA, USA). The scrambled peptide (NRVYNGPAASPVSEQMPIN), the corisin (IVMPESSGNPNAVNPAGYR), and the corisin-like peptide (IVMPESGGNPNAVNPAGYR) were synthesized and provided by Peptide Institute Incorporation (Osaka, Japan). Transforming growth factor (TGF)β1 was purchased from the R&D System (Minneapolis, MN, USA).