Rheumatoid arthritis synovial fibroblasts exhibit acute endoplasmic reticulum stress. (a) Synovial fibroblasts from patients with rheumatoid arthritis (RA) were stimulated or not with 10 ng/ml TNFα for 72 hours prior to protein isolation. After fractionation of the lysates by SDS-PAGE, they were immunoblotted and probed for expression of phosphorylated eukaryotic initiation factor 2α (peIF2α), activation of transcription factor 6 (ATF6), Bip or tubulin. Results are representative of at least three different RA lines. (b) Cells were cultured for 72 hours with or without the addition of 10 ng/ml TNFα or 2 μg/ml tunicamycin. RNA was isolated, reverse-transcribed and amplified for Xbp1, Edem1, CHOP or actin. Xbp1 amplification products were subsequently subjected to Pst1 cleavage before analysis on agarose gels. Results shown are representative of four different RA lines. (c) Fibroblasts were treated with 10 ng/ml TNFα or were left untreated for 24 or 72 hours prior to the isolation of cellular lysates. Following SDS-PAGE and immunoblotting, band intensities of peIF2α were determined and normalized to tubulin levels to compensate for loading differences. The normalized peIF2α levels of TNFα-stimulated cells were then compared with the nonstimulated control. 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 in TNFα-stimulated cultures compared with non-induced cultures: *P < 0.05. NS not significant.