Aprotinin

Cells were extracted with cell lysis buffer [50 mM Tris (pH 7.6), 150 mM NaCl, 1% TritonX-100, 1% deoxycholate, 0.1% SDS, 1 mM PMSF, and 0.2% Aprotinin (Beyotime)]. After measuring protein concentration by BCA protein assay (Beyotime), equal protein amounts were mixed with 5×sample buffer (Beyotime) and boiled. These samples were resolved on 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride) membrane by using semi-dry transfer method. After blocking in 10% nonfat dry milk in TBST (tris-buffered saline tween-20) for 2 h, blots were incubated with primary antibodies including Col II (rabbit polyclonal 1:500, Abcam), Sox9 (rabbit polyclonal 1:500, Abcam), Runx2 (rabbit polyclonal 1:500, Abcam), Col X (rabbit polyclonal 1:500, Abcam) and GAPDH (rabbit polyclonal 1:1,000, Abcam) at 4℃ overnight. After washing with TBST three times, blots were incubated with goat anti-rabbit secondary antibody (1:2,000, ZSGB-BIO) at room temperature for 1 h. GAPDH was used as loading control.

The cells were extracted with lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM PMSF and 0.2% Aprotinin (Beyotime). After we measured the protein concentration, the equal protein samples were mixed with 5X sample buffer (Beyotime) and boiled. The proteins (30 µg) were resolved by 10% SDS-PAGE gel and transferred on PVDF membrane (Millipore, Hong Kong, China) by using the semi-dry transfer method. After blocking in 10% nonfat dried milk in TBST for 2 h, the blots were incubated with primary antibodies including HDAC4 (rabbit polyclonal 1:500, ab12172), p38 (rabbit polyclonal 1:500, ab27986), Runx2 (rabbit polyclonal 1:500, ab23981), Col10a1 (rabbit polyclonal 1:500, ab58632) (all from Abcam), p-p38 (rabbit polyclonal 1:500, bs-50486R), Mmp13 (rabbit polyclonal 1:500, bs-0575R) (both from Bioss) and GAPDH (rabbit polyclonal 1:1,000, ab37168; Abcam) at 4°C overnight. After washing by TBST, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1:2,000, sc-2374; Santa Cruz Biotechnology) at room temperature for 1 h. Blots against GAPDH served as loading control.

p53 protein expression was assessed by western blotting. The cells were lysed in radioimmunoprecipitation assay buffer containing 150 mM NaCl, 50 mM Tris-Cl (pH 7.6), 5 mM EDTA, 0.5% NP-40, 0.5% Triton X-100 containing 10 µg/ml each of leupeptin, aprotinin, and antipain, 1 mM sodium orthovanadate and 0.5 mM phenylmethanesulfonyl fluoride (all Beyotime Institute of Biotechnology, Shanghai, China). The protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Total protein (50 µg) was loaded onto each lane, separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After being blocked with 5% skimmed milk in Tris-buffered saline (pH 7.6; Beyotime Institute of Biotechnology) at room temperature, the membrane was incubated at 4°C overnight with rabbit anti-p53 (1:1,000) and anti-β-actin (1:1,000) primary antibodies. Following incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000), the immune complexes were detected by enhanced chemiluminescence western blotting reagents (Beyotime Institute of Biotechnology). Data were normalized to β-actin protein expression levels and relative protein expression levels were determined using AlphaView Stand Alone Analysis Software 3.0 (ProteinSimple, San Jose, CA, USA).