Ab91655

After 21 d of osteogenic differentiation, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS. Cells were then permeabilized with 0.1% Triton-X (Sigma, USA) for 15 min and blocked with 5% BSA for 1 h (Beyotime, China) at room temperature. Cells were incubated overnight at 4°C with the following primary antibodies: OCN (sc30045, Santa Cruz Biotechnology) and OPN (ab91655, Abcam). Cells were then washed with PBS and incubated with a biotin-labeled secondary antibody (ABclonal, China) for 1 h at room temperature. Cells were then stained with diaminobenzidine (DAB) (Beyotime, China) to detect the protein and then visualized using a microscope (Olympus BX51, Japan).

After 14 days of the corresponding treatment, C3H10T1/2 cells were fixed with 4% paraformaldehyde and washed with PBS. The cells were permeabilized with 0.1% Triton-X (Sigma, USA) and blocked with 10% BSA (Beyotime, China) to reduce non-specific staining. Subsequently, the cells were incubated with anti-osteocalcin (OCN) antibody (sc30045, Santa Cruz Biotechnology) and anti-osteopontin (OPN) antibody (ab91655, Abcam) at 4 °C overnight. The cells were washed three times with DPBS and incubated with a biotin-labelled secondary antibody (Santa Cruz Biotechnology, USA) for 20 min at 37 °C. The cells were washed three times with DPBS, and a streptavidin-HRP conjugate was added to the cells to incubate for 20 min at 37 °C. The presence of the expected protein was observed by diaminobenzidine (DAB) staining and examined under a microscope. Control IgG staining was used as a negative control.

Western blot was used to analyze the protein expression levels of COL1, RUNX2, OPN and OSX. The hPDLCs were treated as described for the qRT-PCR assay. The cells were disrupted in pre-cooled RIPA buffer (Beyotime) after being treated with EGCG for 14 d. The protein content in the extracted cell lysates was determined using a Pierce™ BCA Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. The obtained proteins (20 μg) were separated by 8~12% SDS-polyacrylamide gels. Then, the proteins were transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, MA, USA) with a pore size of 0.45 μm, followed by blocking with 5% fat-free dry milk for 3 h. Subsequently, the membranes were incubated with individual primary antibodies at 4 °C overnight. The following antibodies were used: anti-COL1 (ab138492, Abcam, MA, USA), anti-RUNX2 (ab23981, Abcam), anti-OPN (ab91655, Abcam) and anti-OSX (ab22552, Abcam). The membranes were then incubated with the secondary antibody (ab205718, Abcam) conjugated with horseradish peroxidase for 1 h. The protein bands on the PVDF membranes were then stained using an enhanced chemiluminescence reagent. The stained bands were visualized using the Image Studio Lite software (Media Cybernetics Inc., Bethesda, MD, USA) and quantified by comparing the band intensities to that of GAPDH.