Collagen 1a

The cells were seeded at a density of 4 × 10⁴ cells in 500 μL simple osteogenic medium/well in Nunc Lab-Tek 4 wells chamber slides. After 10 days of cultivation, the cells were washed 3 times with Phosphate Buffered Saline (PBS) and fixed with 4% paraformaldehyde solution for 20 min at room temperature. The cell permeabilization was performed with 0.1% Triton X-100 for 20 min, followed by blocking of unspecific antibody bound with 10% Bovine Serum Albumin (BSA) for 20 min at room temperature. Staining with primary antibodies was performed for bone cells markers: collagen 1A, osteopontin, and osteocalcin (all from Santa Cruz Biotechnologies) at a dilution of 1:50. Samples were incubated overnight at 4 °C in dark, followed by incubation 45 min at room temperature with secondary antibody goat anti-mouse IgG labeled with FITC (Santa Cruz Biotechnologies) (1:50). Slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnologies) for nuclei staining. Each step was followed by 3 washes with PBS. The samples were visualized with a Zeiss Axiovert fluorescence microscope using filters at 488, 546 and 340/360 and images were taken with an AxioCam MRC camera.

Proteins were extracted from each group of left ventricular tissues with lysis buffer supplemented with protease inhibitor and phenylmethylsulfonyl fluoride (PMSF). Proteins (20 μg /sample) were separated by electrophoresis with 10% SDS/PAGE gels. After electrophoresis, proteins were transferred to nitrocellulose membranes. The blots were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with primary antibodies against mouse LOX-1 (a gift from Dr. Sawamura; 1:2,000, v/v, dilution), fibronectin (Abcam; 1:2,000, v/v, dilution), collagen-1a (Santa Cruz, Dallas, TX, USA; 1:1,000, v/v, dilution), collagen-3a (Santa Cruz; 1:1,000, v/v, dilution), collagen-4a (Santa Cruz; 1:1,000, v/v, dilution), p22^phox (Santa Cruz; 1:1,000, v/v, dilution), gp91^phox (Abcam; 1:2,000, v/v, dilution) or β-actin (Santa Cruz; 1:2,000, v/v, dilution) in TBS-T at 4°C overnight on a shaker. After washing with TBS-T three times, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots were scanned with a ChemiDOC XRS system (Bio-Rad, Hercules, CA, USA) following exposure to Luminol Reagents (Beyotime, Shanghai, China) for 3 min.

hUCB-MSCs were obtained from MEDIPOST Co. Ltd (Seoul, Korea). Fetal bovine serum was bought from BioWhittaker Inc. (Walkersville, MO, USA). AA, A23187, bisindolylmaleimide I, BrdU, Indomethacin, LY294002, mitomycin C, Nordihydroguaiaretic acid (NDGA), rapamycin, 1-Aminobenzotriazole (1-ABT), and SB203580 were aquired from the Sigma Chemical Company (St Louis, MO, USA). Phospho-Akt^ser473, phospho-Akt^thr308, Akt1/2/3, β-Actin, collagen1A, collagen3A, collagen5A, fibronectin, phospho-p38, p38, phospho-JNK, JNK, p-ERK1/2, ERK, lamin A/C, MMP-12, pan-cadherin, PKCα, PKCɛ, PKCθ, PKCζ, PKC, Phospho-PKCζ, and Sp1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-PKC, phospho-mTOR^ser2481 (mTORC2), phospho-mTOR^ser2448 (mTORC1), and mTOR were purchased from Cell Signaling (Beverly, MA, USA). The Akt inhibitor I was aquired from Calbiochem (La Jolla, CA, USA). The CD34, GPR40, phospho-Sp1, and MT3-MMP antibodies were obtained from Abcam (Cambridge, MA, USA). Mithramycin A was purchased from Tocris (Bristol, UK). Zymogram gels were bought from Novex (San Diego, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA, USA). All other reagents were used as received and were of the highest purity commercially available.