FIGURE 6.

Silencing of Ccdc80 by RNAi impairs repression of TCF-mediated transcription and Wnt/β-catenin target genes expression during adipocyte differentiation. A-D, stable 3T3-L1 cell lines transduced with retrovirus encoding a non-silencing (NS) shRNA (white bars) or an shRNA against mouse Ccdc80 (black bars) were created. Expression of Wnt 10b (A) and Wnt/β-catenin targets (D) (Axin-2, cyclin D1 and Frizzled-7). Gene expression was determined by real-time PCR using a low density array. ^*, p < 0.05 versus NS shRNA. TOPFLASH activity (B) and β-catenin protein expression (C) were determined in postconfluent cells (Day 0) and after the addition of adipogenic inducers (dexamethasone, IBMX, and insulin) for 1, 2, and 4 days. ^*, p < 0.05 versus NS shRNA. E, 3T3-L1 cells were infected with adenovirus encoding either LacZ (Ad-LacZ, white bars) or mouse Ccdc80 (Ad-Ccdc80, black bar) at a m.o.i. of 500, 1000, and 2000. TOPFLASH activity was determined in postconfluent cells (Day 0) and after the addition of adipogenic inducers (dexamethasone, IBMX, and insulin) for 4 days. F, cyclin D1 repression during clonal expansion. Cyclin D1 expression was determined by real-time PCR in 3T3-L1 stably transduced with retrovirus encoding a NS shRNA (white bars) or an shRNA against mouse Ccdc80 (black bars) (knockdown; left portion of the graph) or in 3T3-L1 infected with adenovirus encoding either LacZ (Ad-LacZ, white bars) or mouse Ccdc80 (Ad-Ccdc80, black bar) at a m.o.i. of 2000 (overexpression; right portion of the graph). Data are presented as % change in cyclin D1 expression during differentiation (Day 0 to Day 4).