Fig. 5. (A) Expression of Pou4f2 in HEK293 cells stably transfected either with a Wt1(–KTS) expression construct or with the empty pCB6⁺ vector. Pou4f2 mRNA levels were quantified by real-time RT–PCR using the light cycler system (Roche Molecular Biochemicals) and normalized for GAPDH transcripts. Shown are the data obtained from four independent clones each of pCB6⁺ and Wt1(–KTS) transfected cells. Note that stable expression of Wt1(–KTS) increased Pou4f2 mRNA levels in HEK293 cells ∼8-fold. The horizontal bars indicate the mean values in each group. (B) Relative luciferase activities measured in the lysates of U2OS human osteosarcoma cells. U2OS cells were transiently co-transfected with phBrn3bUS3.8, Wt1 expression constructs encoding two different splice variants (+KTS/–KTS isoforms), and a cytomegalovirus-promoter driven β-galactosidase expression vector that was used for normalization of transfection efficiencies. Plasmid phBrn3bUS3.8 contained an ∼3.8 kb EcoRV–XhoI genomic sequence from the 5′-regulatory region of the human Pou4f2 gene (schematic drawing) in the pGL2 basic reporter vector. Values shown are means ± SEM of n = 7 experiments each performed in duplicate. P <0.05 was considered statistically significant (ANOVA).