(A). Mutation of Sp1 sites decreases activation of the cyclin D3 promoter. Three mutation constructs were made in the context of the full promoter (pD3GH) as follows: pD3-m1, pD3-m2, and pD3-m1/m2, which contain mutations in the Sp1 site at −64 to −72, at −121 to −129, or in both Sp1 sites, respectively. The nucleotides mutated in each Sp1 site are as described in the legend to Fig 5. The mutation constructs were transfected into Y10 cells, which were then cultured in the presence or absence of MGDF and assayed for HGH production, as detailed in Materials and Methods. All experiments were normalized for transfection efficiency by cotransfection with pCMVβ-galactosidase, as described in Materials and Methods. The data are the means of four determinations with standard deviations indicated by the error bars. (B) Sp1 binding-ability, cylin D3 promoter activity, and cyclin D3 levels in cells cultured with MGDF for different times. Sp1 binding ability to the fragment –190/−99 of the cyclin D3 promoter was determined by EMSA, as in Fig 5. Cyclin D3 promoter activity (pD3GH) and cyclin D3 protein level were determined as in Figs 2 and 1, respectively. The data shown are of a representative experiment out of two performed. The intensities of the bands (in EMSA and Western blots) were quantitated using the Electrophoresis Documentation and Analysis System.