Figure 6
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-1β 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-1β for different times. Cell lysate was prepared in RIPA buffer and analyzed for COX-2 expression by western blotting. β-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-1β 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-1β for different times. Cell lysate was prepared in RIPA buffer and analyzed for COX-2 expression by western blotting. β-actin is used as loading control.
