Figure 7

Proteasome inhibition affects expression of autophagy markers. (a) Rheumatoid arthritis (RA) synovial fibroblasts were cultured with 2 μg/ml tunicamycin, 0.5 μM epoxomicin, 12.5 μM chloroquine (CQ) or 10 mM 3-methyladenine (3-MA) for 1 hour prior to the addition or not of 10 ng/ml TNFα for 24 hours. Cellular lysates were immunoblotted and probed for microtubule-associated protein 1 light chain 3 (LC3) or tubulin as a loading control. Band intensities were quantified using ImageJ software. (b) The total amount of LC3 in the samples relative to that in the TNFα sample was determined. Significant differences in TNFα-stimulated cultures compared with cultures where a proteasome inhibitor was included in addition to TNFα: **P < 0.01. (c) The total amount of p62 in the samples relative to that in the TNFα sample was determined. Significant differences in TNFα-stimulated cultures compared with cultures where a proteasome inhibitor was included in addition to TNFα: *P < 0.05. (d) LC3 results from (a) are plotted to show the relationship between total LC3 and LC3-II relative to total LC3: (i) without TNFα, (ii) with TNFα, (iii) samples treated with proteasome inhibitor or macroautophagy inhibitor in the presence of TNFα shown in (ii) replotted with the samples without TNFα treatment shown in (i) to show the restoration of the linear relationship between total LC3 and LC3-II relative to total LC3. This suggests that the decreased LC3 levels observed in the presence of TNFα result from proteasome activity as well as macroautophagy. In all cases, results are representative of at least three different experiments.