Lysosome or proteasome inhibition results in endoplasmic reticulum stress. (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. A representative blot probed for expression of phosphorylated eukaryotic initiation factor 2 alpha (peIF2α) or eukaryotic initiation factor 2 alpha (eIF2α) is shown. Blots were scanned and analyzed by ImageJ software. (b) The relative amount of phosphorylated eIF2α in cells stimulated with 12.5 μM CQ, 10 mM 3-MA, 0.5 μM epoxomicin or 2 μg/ml tunicmycin in addition to TNFα was determined using tubulin as a loading control. These were then compared with the peIF2α levels in TNFα-stimulated control cells. Significant differences in TNFα-stimulated cultures compared with cultures where the autophagy inhibitors CQ or 3-MAaproteasome inhibitor (PI) or tunicamycin were included in addition to TNFα: *P < 0.05, **P < 0.01. (c) The effect of 12.5 μM CQ, 10 mM 3-MA, 0.5 μM epoxomicin or 2 μg/ml tunicmycin in addition to TNFα on the expression of the cleaved/active form of ATF6 was determined. Blot shown is representative of at least three different lines. (d) CHOP mRNA levels were determined in cells that had been stimulated for 24 hours with the inhibitors as indicated. They were compared with nonstimulated cells. The mean and standard deviation of CHOP expression in three different lines is shown. Significant differences in TNFα-stimulated cultures compared with cultures where PI or tunicamycin was included in addition to TNFα: *P < 0.05.