Cells were lysed and extracted using lysis and extraction buffer (Pierce IP Lysis Buffer; cat. no. 87787; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols on day 7(24 (link)). Protein in the whole-cell lysates was quantified using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). A total of 10 µg/lane of protein samples were loaded on a 7.5% gel for collagen I and loaded on a 10% gel for RUNX2 and GAPDH experiments, respectively and then transferred to polyvinylidene difluoride membranes (Immun-Blot®; Bio-Rad Laboratories, Inc.) for immunoblotting. The membranes were blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4˚C: Anti-collagen I (1:500; cat. no. ab6308; Abcam), anti-RUNX2 antibody (1:200; cat. no. ab76956; Abcam) and anti-GAPDH antibody (1:2,000; cat. no. ab9485; Abcam). After washing with TBS-0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, goat anti-mouse immunoglobulin G (IgG; cat. no. ab205719; Abcam) and goat anti-rabbit IgG (cat. no. ab205718; Abcam) at 1:10,000 dilution for 2 h at room temperature. The immunoblot signals were visualized using horseradish peroxidase substrate (cat. no. WBKLS0100; Merck KGaA).