Figure 3

Regulation of nitric oxide and inducible nitric oxide synthase mRNA expression by transduced Tat-SOD. (a) After monolayer cultured chondrocytes were transduced with 1 to 7 μM of Tat-SOD, control SOD or Tat-GFP proteins, serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Culture media were harvested after 24 hours, and the production of nitric oxide(NO) was analyzed using a nitrate/nitrite colorimetric assay kit. The data are means and standard deviations of duplicate experiments from samples from three different donors. Asterisks denote p < 0.05 compared to control (IL-1 treatment alone). (b) Explant cultured chondrocytes were transduced with 1 to 7 μM of Tat-SOD, control SOD or Tat-GFP proteins, and serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Culture media were harvested after 72 hours, and the production of NO was analyzed using a nitrate/nitrite colorimetric assay kit. The data are means and standard deviations of duplicate experiments from samples from three different donors. Asterisks denote p < 0.05 compared to control (IL-1 treatment alone). (c) Regulation of IL-1 stimulated NO production in chondrocytes by transduced Tat-GFP was observed in monolayer (left panel) and explant (right panel) cultured chondrocytes. Chondrocytes were transduced with 1 to 7 μM of Tat-GFP, and serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Culture media were harvested after 24 hours for monolayer or 72 hours for explants, and the production of NO was analyzed using a nitrate/nitrite colorimetric assay kit. The data are means and standard deviations of duplicate experiments from samples from three different donors. (d) After transduction of chondrocytes with 3 μM of each fusion protein, serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Total RNA was isolated from chondrocytes after 4 hours and RT-PCR was performed. Data are representative of four samples from different donors. The band densities for iNOS mRNA were quantified, the percent GAPDH density was calculated for iNOS and the value for the control culture was set at 1. Asterisks denote p < 0.05.