FIGURE 8.

UGDH-specific siRNA down-regulates UGDH mRNA, protein, and enzyme activity levels in orbital fibroblasts. A, cultures reaching 80% confluence were transfected with scrambled (Control) or UGDH-specific siRNA using Oligofect reagent. Cells were disrupted, and cellular RNA was analyzed by real-time PCR. Data are expressed as the mean ± S.D., n = 3 independent determinations; **, p < 0.01 versus control siRNA. In all experiments, UGDH siRNA reduced UGDH levels by 86% (p < 0.01 versus control siRNA). B, UGDH protein levels were substantially reduced in orbital fibroblasts after transfection with UGDH-specific siRNA. Cells were transfected as described under “Experimental Procedures” and lysed, and cellular proteins were subjected to Western blot analysis with an anti-UGDH Ab. C, cultures were transfected with scrambled (Control) siRNA or UGDH-specific siRNA and lysed, and UGDH enzyme activity was assayed in the absence or presence of UDP-Glc as described under “Experimental Procedures.” *, p < 0.05. In three separate experiments, UGDH siRNA reduced UGDH activity by 41 ± 4% (p < 0.001 versus control). D, orbital and dermal fibroblasts were transfected with scrambled (Control) or Sp1-specific siRNA, cell layers were assayed for UGDH activity in the absence or presence of UDP-Glc. Data are expressed as the mean ± S.D. of triplicate determinants from one experiment. **, p < 0.01 versus control siRNA. When results of separate experiments were combined, Sp1 siRNA reduced activity in orbital fibroblasts by 40 ± 4% versus controls.