Fig. 2. Proliferation capacity and transcriptional transactivation from Tfc/Lef1 promoters in homozygous, heterozygous and ß-catenin-null mutant keratinocytes. (A) Proliferation curves were established under low Ca²⁺ conditions for the indicated keratinocyte cultures. Error bars represent standard deviation of the mean of triplicate samples within one out of two experiments. (B) During the proliferative phase (in A, 96 hours), cyclin D1 expression was simultaneously assessed by western blot analysis. Plakoglobin expression examined on the same blot served as a control for equal loading. (C) Keratinocytes were transfected with pTOP-flash (gray bars) or the same amount of pFOP-flash (open bars) reporter along with Lef1 or ß-catenin as indicated. Relative light units (firefly luciferase over renilla luciferase) are indicated. Numbers are the ratios of normalized TOP-flash over FOP-flash activity. Bars represent minimum and maximum values of duplicate samples of one experiment. This experiment was repeated three times. (D) Expression of Tcf/Lef family members assessed by RT-PCR. Total RNA was isolated from confluent cell cultures grown under low (1) or high (h) Ca²⁺ conditions. Plasmids, full-length cDNA as positive control; DNA, genomic DNA used as negative control; H₂O, PCR performed without template. For Tcf-3 and GAPDH, the number of cycles was reduced to 25 and 15, respectively, to allow semi-1uantitative analysis.