Figure 3
COX-2 protein expression correlates with the assembly and disassembly of SGs in OA chondrocytes.
(a) Human OA chondrocytes were seeded in 35-mm dishes (1 × 106 OA chondrocytes/dish) for 2 to 3 days and then treated with IL-1β for the indicated times. Total RNA was isolated and the COX-2 transcript level was measured by TaqMan assay for COX-2. The graph shows fold change of COX-2 mRNA relative to β-Actin. The data represent the relative levels of COX-2 mRNA from 3 patient samples (mean ± SD). (b) OA chondrocytes were seeded in 35-mm dish as above and treated with IL-1β for the indicated times, harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor. The lysate was analyzed for GRP78, COX-2, eIF2α phosphorylation and stress granule marker proteins by Western blot. (c) and (d) Densitometric analysis of COX-2 (relative to β-actin) and phospho-eIF2α (relative to eIF2α) was performed using ImageJ. The data represent the relative levels of COX-2 protein or phospho-eIF2α from 3 patient samples with the standard deviation. (e) OA chondrocytes were seeded in 8-well chamber slides as described in Fig. 1. The OA chondrocytes were treated with IL-1β for the indicated times, fixed with 4% PFA and permeabilized with 0.3% Triton X100-PBS. The formation of SGs was analyzed by immunofluorescence staining with anti-G3BP1 antibody.
