Tunel assay kit

The apoptotic ATDC5 cells were stained using TUNEL assay kit (KeyGen, Biotechnology, Co., LTD, China) according to the manufacturer's protocol. Brie y, cells seeded on the slides were xed with 4% paraformaldehyde at room temperature for 0.5 h. After treatment with 1% Triton X-100/10 mM PBS for 5 minutes, proteinase K was added. Then cells were incubated with TdT enzyme reaction solution, streptavidin-HRP working solution and DAB staining working solution in the dark, respectively. Hematoxylin was used to stain the nucleus.
Western blot analysis ATDC5 cells were homogenized in RIPA lysis buffer containing protease inhibitors (Roche, China) for 30 min, then centrifuged at 4 °C to separate the lysates. The concentration and purity were determined by BCA kit (Solarbio, China). Next, 30 mg of protein was transferred onto PVDF membranes (Millipore, USA) and blocked with non-fat milk at room temperature for 2 h. The membrane was then incubated with primary antibodies against SFRP5, Bcl-2, Bax, Caspase 3, Wnt5a, JNK, phosphorylated (p)-JNK, and GAPDH (Santa Cruz Biotechnology, USA). The secondary antibodies were conjugated with horseradish peroxidase (Santa Cruz) and incubated for immunoblotting analysis. Signal detection was measured using an ECL Western blotting detection kit (Amersham Biosciences, USA). The expression of the target gene was represented relative to GAPDH.