Figure 2.

TNFα affects autophagy. Lysates from fibroblasts stimulated or not with 10 ng/ml TNFα, 12.5 μM chloroquine (CQ) or 4 mM 3-methyladenine (3-MA) for 24 or 72 hours were analyzed by immunoblotting. (a) A representative immunoblot probed with antibodies to microtubule-associated protein 1 light chain 3 (LC3), p62 or tubulin. Results are representative of at least three different rheumatoid arthritis (RA) lines. (b) The amount of total LC3 was determined from quantification of LC3-I and LC3-II. Protein loading was corrected by quantification of tubulin. The ratio of total LC3 in TNFα-stimulated cells is compared with their nonstimulated controls. Filled circles, RA lines at 24 hours; open circles, RA at 72 hours; filled squares, control at 24 hours; open squares, control at 72 hours. Significant differences between TNFα-stimulated cultures compared with either non-induced cultures or with cultures that included 3-MA or CQ in addition to TNFα: *P < 0.05, **P < 0.01. (c) The ratio of LC3-II to total LC3 was determined by quantification of the band intensities using ImageJ software. Filled circles, RA synovial fibroblasts at 24 hours; open circles, RA synovial fibroblasts at 72 hours; filled squares, control fibroblasts at 24 hours; open squares, control fibroblasts at 72 hours. Significant differences between TNFα-stimulated cultures compared with either non-induced cultures or with cultures that included CQ in addition to TNFα: *P < 0.05, **P < 0.01, ***P < 0.001. (d) Fibroblasts grown on chamber slides for 24 hours with or without 10 ng/ml TNFα were examined by fluorescence microscopy for LC3. Nuclei were visualized with DAPI. Results are representative of three lines. Ctl, control.