3 methyladenine 3 ma

The chondrocytes were seeded and cultured in 6‐well plates for 4 days before treatment with TM (0.5 μg/ml). Following treatment for 0, 6, 12, and 24 h, the apoptosis ratio of the cells was quantified using an Annexin V‑FITC flow cytometric kit (KeyGen Biotech. Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Briefly, the cells were harvested and washed in PBS three times. Next, the cells were suspended in 500 μl of ice‐cold binding buffer and incubated with 5 μl of Annexin V‑FITC solution and 5 μl of propidium iodide (PI) solution for 30 min. Finally, the apoptosis ratio of the cells was measured using a flow cytometer (BD Biosciences, San Jose, CA, USA). In addition, the apoptotic ratio of chondrocytes pre‐treated with 3‐methyladenine (3‐MA, 2 mM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h was quantified after treatment with TM (0.5 μg/ml).

Rabbit mAb to LC3I antibody (12741, dilution 1/800), rabbit mAb to p62 (23214, dilution 1/1000), rabbit anti-Beclin-1 (3495, diluted 1/1000), rabbit anti-Phospho-ULK1 (14202, diluted 1/800) and ULK1 (8054, diluted 1/1000) were all purchased from Cell Signaling Technology, Inc. Rabbit polyclonal anti-LC3II antibody (ab63817, dilution 1/1000) was purchased from Abcam. Anti-actin was obtained from Epitomics, an Abcam company (Cambridge, MA, USA). miR-34a inhibitors (5’-AGCCUUGCUGCAGGUGCGCAU-3’) and a nonsense control (NC) sequence (5’-UGCCUUACUGACGGUCGGAGA-3’) were obtained from Shanghai GeneChem, Inc. (Shanghai, China). According to the protocol, Huh7-R and HepG2-R cells (1 × 10⁵) were transfected with 50 nM miR-34a inhibitors, 50 nM miR-34a mimics, 50 nM scramble control inhibitors or 50 nM scramble control mimics using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) for ~ 30 min at room temperature. After 48 h of transfection, the transfected cells were used for subsequent experiments. 3-Methyladenine (3-MA), a class III phosphoinositol 3-kinase (PI3K) inhibitor, and choroquine (CQ), were used as selective inhibitors of autophagy, was purchased from Santa Cruz Biotechnology (Texas, U.S.A). Rapamycin (rapa), a potent and specific mTOR inhibitor used as an autophagy agonist, was obtained from Selleck.