Cell protein extraction reagent

The BMSCs were lysed using a cell protein extraction reagent (KEYGEN Biotech, Nanjing, China), and then mixed with loading buffer at a ratio of 4:1 (v/v) before being boiled for 5 min. The proteins were separated using 10% SDS-PAGE and subsequently transferred onto a PVDF membrane. The membrane was then blocked with blocking buffer (Thermo Fisher Scientific, MA, USA) for 10 min and incubated with the following antibodies (Table S3) overnight at 4 °C. On the second day, the bands were incubated with a secondary anti-rabbit antibody (1:3 000 BEYOTIME, Shanghai, China) for 1 h after washing with TBST. The results were visualized using an ECL chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

Exposed to 250 nmol/L TDNs for 1, 3 and 7 days, respectively, the PDLSCs samples were rinsed with PBS softly. The cell protein extraction reagent (KeyGen Biotech) was used to harvest the total proteins. In order to make these collected proteins denature and depolymerize, the 1/4 (v/v) ratio of loading buffer was added and the mix was boiled for 4‐5 minutes. After that, the 10% SDS‐PAGE was performed to separate total albumens into different molecular weight albumens. Subsequently, the target albumens that included the osteogenic differentiation proteins, such as RUNX 2, OPN, and the Wnt signalling pathway‐related proteins, such as β‐catenin, LEF 1 and GSK‐3β, were transferred to a polyvinylidene fluoride membrane. Then the membranes containing these target proteins were immersed in the mixture solutions that included 2.5% bovine serum albumin and 2.5% skimmed milk powder for about 50 minutes to block albumens and incubated overnight with primary antibodies (Abcam) at 4°C. Next day, after rewarming for 60 minutes at 37°C, these membranes were washed thrice with TBST and incubated with secondary antibodies (Beyotime, Shanghai, China) for 1 hour. After being washed with TBST, the bands of albumens were visualized by an ECL chemiluminescence detection system (Bio‐Rad, Hercules, CA, USA). The housekeeping control protein was GAPDH.

The treated ASCs were rinsed with cold PBS three times, and total proteins were harvested with a cell protein extraction reagent (KEYGEN Biotech, Nanjing, China). The collected proteins were then mixed with loading buffer at a ratio of 4:1 (V/V) and boiled for 5 minutes. The proteins were separated on a 10% SDS‐PAGE, and proteins were subsequently transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 1 hour at 37°C, the membranes were incubated with primary rabbit monoclonal antibodies specific to the following targets: OPN, Runx2, Lef‐1, GSK, cyclin D and β‐catenin (Abcam, Cambridge, UK). Then, each band was incubated with a secondary labelled anti‐rabbit antibody (BEYOTIME, Shanghai, China), and the results were visualized using an ECL chemiluminescence detection system (Bio‐Rad, Hercules, CA, USA).