Figure 1
IL-1β induces the assembly of stress granules in human OA chondrocytes.
(a) Primary human OA chondrocytes isolated from OA patients were seeded in 35-mm dishes (1 × 106 OA chondrocytes/dish) for 2 to 3 days in DMEM-F12 supplemented with 10% serum. OA chondrocytes were either treated or untreated with IL-1β (10 ng/ml) and harvested after 24 hours. Cell lysate was prepared in RIPA buffer supplemented with protease and phosphatase inhibitor and expression of COX-2 and GRP78 (BiP) was analyzed by Western blotting. β-actin was used as a loading control. (b) OA chondrocytes were treated with IL-1β for 1 hour. The OA chondrocytes were lysed and analyzed for eIF2α phosphorylation. Total eIF2α was used as loading control. (c,d) Primary OA chondrocytes were seeded in 8-well chamber slides (0.05 × 106 OA chondrocytes/well) for 2 to 3 days. The OA chondrocytes were treated with IL-1β for 1 hour and fixed with 4% paraformaldehyde (PFA) and permeabilzed in 0.3% Triton X-100. SGs were visualized by immunofluorescence staining with primary antibody specific to TIA-1 followed by Alexafluor 546 secondary antibody. The quantification of SGs is shown in Fig. 1d. (e) Cells were treated with IL-1β as above and probed with G3BP1, Staufen-1 and HuR followed by Alexafluor 594 secondary antibody to analyze SGs assembly. (f) OA chondrocytes were transfected with a G3BP1-GFP plasmid construct for 48 hours followed by treatment with Il-1β or H2O2 for 1 hour. The OA chondrocytes were fixed as above and counter-stained with DAPI to visualize the nuclei and mounted with anti-fade mounting medium. The slides were visualized with an Olympus FV1000 confocal microscope.
