Reduced cell proliferation by Wnt pathway is β-catenin-dependent but Tcf3-independent.

<p><b>(A)</b> qRT-PCR for Wnt targets (<i>Axin2</i>, <i>Sp5</i>, <i>CyclinD</i>, <i>C-Myc</i>), Tcf1-binding cell cycle genes (<i>p15</i>^<i>Ink4b</i>, <i>p16</i>^<i>Ink4a</i>, <i>p18</i>^<i>Ink4c</i>, <i>p19</i>^<i>Arf</i><i>)</i> in control and overexpressing β-catenin clones (ESCs-β-cat–OE clones) (n = 3). <b>(B)</b> Representative Western blot of β-catenin, p16^Ink4a, p19^Arf and β-actin in control and ESCs-β-cat–OE clones. <b>(C)</b> Representative images and cell growth curve of control mESCs and three overexpressing β-catenin clones (ESCs-β-cat–OE clones 13, 19 and 27) cultured for 3 passages. <b>(D)</b> Quantitative representation of number of EdU positive cells (EdU+) in control and ESCs-β-cat–OE clones 36h after plating (n = 4). Cells were incubated 45’ with EdU before fixation. Negative control correspond to control cells non-incubated with EdU. <b>(E)</b> Cell number of β-catenin WT (β-catenin^fl/fl) and Knock-out (β-catenin^Δ/Δ) mESCs in serum+LIF grown for 3 days. (n = 4). <b>(F)</b> Cell number quantification of β-catenin^fl/fl and β-catenin^Δ/Δ mESCs treated for 48h with purified Wnt3a or with CHIR99021 at indicated concentrations (n = 4). <b>(G)</b> Representative Western blot of β-catenin, p16^Ink4a, p19^Arf and β-actin in 48h CHIR99021-treated β-catenin^fl/fl and β-catenin^Δ/Δ mESCs. <b>(H)</b> Cell number quantification of WT and Tcf3-/- ESCs treated with BIO or DMSO (0,15%) at the indicated concentrations for 2 and 3 days (n = 3; BIO-treated compared to respective DMSO-treated mESCs). All pooled data are represented as means ± SD. The asterisks indicate statistical significance by two-tailed Student’s t-test analysis (n.s. not significant; * p<0.05; ** p<0.01; ***p<0.001).