Fig. 6. Consequences of pRb and p107 loss in primary keratinocytes. (A) Doubling times of cultured primary keratinocytes showing a reduction in Rb^F19/F19;p107+/–;K14cre and Rb^F19/F19;p107–/–;K14cre with respect to wild-type and pRb-deficient cells. (B) FACS analysis of cell cycle profiles of asynchronous growing keratinocytes showing no significant differences among the different genotypes. Data are from the analysis of four independent experiments. In B, at least 10⁵ cells were scored on each experiment (mean±s.d.). (C) Percentage of BrdU incorporation in keratinocytes growing under low (0.05 mM) and high (1.2 mM) Ca²⁺ medium for the indicated times and re-stimulated with low Ca²⁺ medium. (D) Percentage of BrdU incorporation in primary keratinocytes of the indicated genotypes after adeno cre or adeno GFP infection and before culture under the conditions indicated. Data come from the analysis of three independent experiments scoring at least 1000 cells on each (mean±s.d.). (E) Luciferase reporter activity of E2F of primary keratinocytes of the indicated genotypes cultured in low Ca²⁺ or upon Ca²⁺-induced differentiation for 24 and 48 hours. Transfections were performed in triplicate, and the mean and standard error were calculated for each condition. Two independent transfection experiments were performed and luciferase activity was normalized to the values obtained with control, Rb^F19/F19, cells in low Ca²⁺.