Figure 4
COX-2 mRNAs are sequestered in stress granules.
(a) RNA fluorescence in situ hybridization (FISH) analysis of COX-2 mRNA and co-immunostaining of TIA-1 or HuR. OA chondrocytes were seeded in 8-well chamber slides as described in Fig. 1 and treated with IL-1β for indicated time. RNA FISH was performed as per the manufacturer’s protocol (RNAscope assays, Advanced Cell Diagnostics). After RNA FISH, the OA chondrocytes were probed with anti-TIA-1 or anti-HuR antibody to visualize SGs. The OA chondrocytes were mounted with antifade mounting medium containing DAPI and images were acquired. (b) OA chondrocytes (50 × 106 OA chondrocytes) were treated with IL-1β for 2 hours, washed extensively with PBS and lysed in polysome lysis buffer to immunoprecipitate large RNP complexes. The lysate was immunoprecipitated with rabbit-G3BP1, rabbit-HuR, rabbit-Satufen-1, mouse-TIA-1 or rabbit- CUGBP2 and mouse or rabbit IgG antibodies as control. The RNA was isolated by Trizol-chloroform extraction and analyzed for COX-2 mRNA by RT-qPCR using a TaqMan assay and each IP was normalized with Ct value from input RNA fraction. The results represent the fold enrichment of COX-2 mRNA from three independent immunoprecipitation experiments. (c) OA chondrocytes were seeded in 35-mm dishes as described earlier. IL-1β was added to the OA chondrocytes for 1 hour followed by the addition of actinomycin D. The OA chondrocytes were harvested at the indicated time post-actinomycin D addition. Total RNA was isolated by Trizol-chloroform extraction and the COX-2 mRNA levels were analyzed by RT-qPCR with a Taqman assay and normalized to β-Actin. (d) OA chondrocytes were treated with IL-1β for 3 hours followed by addition of actinomycin D or Ammonium Chloride for 1 hour. The OA chondrocytes were washed with PBS lysed in RIPA buffer and analyzed for COX-2 by immunoblotting. β-actin is used as loading control.
