Western Blot Analysis
The protocol of Western blot analysis was described previously. In brief, AMDC seeded into 6-well plates at a density of 1 × 105 cells/well and treated by TC with varying concentrations described above (0, 6, 12 μg/mL). After 48 h incubation, total proteins in cells were extracted using radioimmunoprecipitation assay (RIPA buffer high) lysis buffer (60 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS) (Solarbio, R0010, Beijing, China), PMSF 100 Mm (Solarbio, P0100, Beijing, China), Aprotinin from bovine lung (Solarbio, 9087-70-1, Beijing, China), Protein Phosphatase Inhibitor (All-in-one,100x) (Solarbio, P1260, Beijing, China) with the proportion of 100:1:1:1 on ice and quantified using BCA protein concentration determination kit (Beyotime, P0012, Beijing, China) according to the manufacturer’s instruction. Subsequently, proteins were in polyacrylamide gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, NY, USA), and incubated with primary antibodies at 4 °C overnight. After washing with Tris Buffered Saline Tween (TBST; Solarbio, T1086, Beijing, China) for 3 times, the PVDF membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The GAPDH antibody (1:10000; Proteintech, Cat No.:10494-1-AP, Wuhan, China) signal was used as a loading control. Finally, the bound antibodies were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore, NY, USA). Images were obtained with Bio-Rad ChemiDocTM XRS system (Bio-Rad, Shanghai, China). The intensity of the target proteins blots was quantified by Image J software version 1.8.0.