Fig. 2

Determination of autophagy induction by monitoring the conversion of microtubule-associated protein 1 light chain 3 type I (LC3-I) to LC3-II in whole protein extracts derived from osteoarthritis fibroblast-like synovial cells (OA-FLS) and rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). Cells were left untreated or were treated with 0.01, 0.1, 1, or 10 μM methotrexate (MTX) (a) for 48 h or 0.1 μM MTX (b) for the indicated times. Autophagic flux was monitored in RA-FLS after 24 h of treatment with 0.1 μM MTX in the presence or absence of 0.1 μM bafilomycin A1 (Baf) (c). Cells were treated with 0.1 μM MTX for 48 h (d), then harvested and subjected to transmission electron microscopy as described in the Methods section. The results are representative of three experiments using cells from different patients. β-actin was used as a loading control in all experiments. Values in (a) and (b) are the mean ± standard deviation. Student’s t test was used for the data analysis. *p < 0.05 vs. OA-FLS