Fig. 4

Effect of methotrexate (MTX) on autophagy-related proteins. a After the cells were exposed to 0.1 μM MTX for the indicated times, the cell lysates were subjected to Western blotting with specific antibodies. The results are representative of three independent experiments. β-actin was used as a loading control. b The levels of Akt, phosphorylated Akt (p-Akt), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), high mobility group box chromosomal protein 1 (HMGB1), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) proteins were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and corrected for β-actin. Data in columns represent mean of three independent experiments, and values in (b) are the mean ± standard deviation. The Mann–Whitney U test was chosen for analysis of data for the levels of HMGB1 of cells exposed to 0.1 μM MTX for 48 h in Western blot assays, while Student’s t test was used for the rest of the data. *p < 0.05 vs. OA-FLS