Figure 5
Inhibition of autophagy block stress granule’s clearance and dampens COX-2 expression.
OA chondrocytes were seeded in 35-mm dishes as described earlier. The OA chondrocytes were pretreated with either Bafilomycin A1 (a) or Ammonium Chloride (b) for 2 hours followed by the addition of IL-1β for the indicated times. The OA chondrocytes were washed with PBS and lysates were prepared in RIPA buffer supplemented with protease inhibitor and analyzed by western blotting for COX-2 protein. (c) Total RNA was isolated from OA chondrocytes pretreated with Bafilomycin A1 or Ammonium Chloride followed by IL-1β for 6 hours. COX-2 transcripts were quantitated by RT-qPCR with TaqMan assay and normalized to β-Actin. (d) The OA chondrocytes were first treated with IL-1β for 1 hour and then Bafilomycin A1 or Ammonium Chloride was added. The OA chondrocytes were harvested after 6 hours and the lysate was analyzed for COX-2 expression by western blotting. (e,f) The OA chondrocytes were seeded in 8-well chamber slides as described in Fig. 1 and treated with either bafilomycin A1 or Ammonium Chloride for 2 hours followed by IL-1β for 6 hours. The OA chondrocytes were fixed in 4% PFA and immunostained with mouse-TIA-1 and rabbit-G3BP1 primary antibodies and anti-mouse AlexaFluor 546 and anti-rabbit AlexaFluor 594 secondary antibodies. The OA chondrocytes were mounted in antifade mounting medium containing DAPI and images were acquired using an Olympus FV1000 confocal microscope. The graph shows the quantitation of SGs.
