Transduction of transactivator of transcription (Tat)-superoxide dismutase (SOD) fusion protein into explant cultured chondrocytes. Chondrocytes were obtained from a relatively lesion-free area of femoral condyles in OA patients and cultured in explants. (a) Transduction of Tat-SOD into the cells was analyzed by western blotting with a rabbit anti-polyhistidine IgG. Tat-SOD (1 to 7 μM) or control SOD were added to the culture medium for 6 hours. The tissues were milled in liquid nitrogen after transduction and protein was extracted in 4 M guanidine hydrochloride buffer and dialyzed. (b) Immunohistochemistry of cartilage explant transduced with Tat-SOD fusion protein (3 μM concentration of fusion protein added to the culture medium for 6 hours). The explants were fixed and sectioned in a cryotome after extensive washing. Cartilage sections were incubated with a mouse monoclonal anti-human Cu, Zn-SOD IgG and then visualized with a confocal scanning fluorescent microscope. Data are representative of seven samples from different donors. Scale bar = 100 μm.

Transduction of transactivator of transcription (Tat)-superoxide dismutase (SOD) fusion protein into monolayer cultured chondrocytes. Chondrocytes were obtained from the femoral condyle and the tibial plateau from osteoarthritis (OA) patients and cultured in monolayers. First passage chondrocytes were used in subsequent experiments. (a) Dose-dependent and (b) time-dependent transduction of Tat-SOD into chondrocytes. Transduction of Tat-SOD into the cells was analyzed by western blotting with a rabbit anti-polyhistidine IgG. Tat-SOD (1 to 7 μM) and control SOD were added to the culture medium for 1 hour (a), or 3 μM of Tat-SOD and control SOD were added to the culture medium for 1 to 9 hours (b). (c) Localization of transduced Tat-SOD protein. After FITC-labeled Tat-SOD (3 μM) was transduced into chondrocytes, the cells were washed with PBS and immediately observed by fluorescence microscopy (×100 original magnification; inset, ×400 original magnification). Data are representative of four samples from different donors. (d) The specific activities of SOD in cultured chondrocytes treated with Tat-SOD; 1 to 7 μM of Tat-SOD and control SOD were added to the culture medium for 1 hours. Bars represent the mean ± standard error of the mean obtained from duplicate experiments from three donors. Asterisks denote p < 0.05 compared to control.

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.