Fig 2: 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-1ß 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-1ß for 6 hours. COX-2 transcripts were quantitated by RT-qPCR with TaqMan assay and normalized to ß-Actin. (d) The OA chondrocytes were first treated with IL-1ß 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-1ß 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.

Fig 3: Depletion of TIA-1 or HuR expression accelerates COX-2 mRNA translation in OA chondrocytes.(a) OA chondrocytes (1 × 106) were transfected with control or TIA-1 shRNA plasmid constructs. After 48 hours, the OA chondrocytes were harvested and analyzed for TIA-1 knockdown by western blotting using anti-TIA-1 antibody. (b,c) OA chondrocytes were transfected with control or TIA-1 shRNA and seeded in 8-well chamber slides for 48 hours followed by IL-1ß treatment for 1 hour. The OA chondrocytes were washed with PBS, fixed in 4% PFA and immunostained with G3BP1 as described in Fig. 1. (d) OA chondrocytes were transfected with control or TIA-1 shRNA for 48 hours and treated with IL-1ß for different times. Cell lysate was prepared in RIPA buffer and analyzed for COX-2 expression by western blotting. ß-actin is used as loading control. (e) The supernatant from the above experiment was collected and analyzed for PGE2 using an ELISA kit from R&D systems. (f) OA chondrocytes (1 × 106) were transfected with control or HuR siRNA (50 nM) and incubated for 48 hours. The OA chondrocytes were harvested and analyzed for HuR knockdown by western blotting. (g) Chondrocytes were transfected with either control or HuR siRNA for 48 hours followed by treatmetn with IL-1ß for 2 hours. RNA FISH for COX-2 mRNA and protein immunofluorescence staining for TIA-1 was done as in Fig. 4. (h) OA chondrocytes were transfected with control or HuR siRNA for 48 hours and treated with IL-1ß for different times. Cell lysate was prepared in RIPA buffer and analyzed for COX-2 expression by western blotting. ß-actin is used as loading control.

Fig 4: IL-1ß 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-1ß (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. ß-actin was used as a loading control. (b) OA chondrocytes were treated with IL-1ß for 1 hour. The OA chondrocytes were lysed and analyzed for eIF2a phosphorylation. Total eIF2a 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-1ß 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-1ß 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-1ß 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.

Fig 5: Effects of OGT on MAPKs in BV-2 cells. BV-2 were exposed to LPS (1 µg/ml) with or without OGT for 24 h. Whole cell lysates were analyzed for phosphor-extracellular signal-regulated kinase (p-ERK), ERK, phospho-JNK (p-JNK), JNK, phospho-p38 (p-p38), and p38 MAPK expression by immunoblot assay (A). The figures are representative of at least three independent experiments. Immunoblot quantification of p-p38/p38 MAPK are shown in B. BV-2 microglial cells were exposed to LPS (1 µg/ml) for 24 h with SB203580 (20 µM) or OGT (10 µg/ml) (C–E). The level of PGE2 was analyzed by the enzyme immunoassay. COX-1 (D) and COX-2 (E) mRNA were analyzed using real-time qRT-PCR, normalized to that of 18S rRNA and expressed relative to the mean of control. Data are shown as mean ± S.D. of three independent experiments. **P < 0.01 versus control, *P < 0.05 versus control, N.S., not significant.