Fig 3. Activation of canonical Wnt pathway in mESCs reduces cell proliferation and increases the number of cells in G1 phase.
(A) Representative images and quantification of cell number for mESCs treated with purified Wnt3a for 2 and 3 days at the indicated concentrations (n = 3). Scale bar: 400μm. (B) Representative images and cell number quantification for mESCs treated with BIO (μM) at indicated concentrations and with DMSO (μl/ml), as control, for 1, 2 and 3 days (n = 3; BIO-treated compared to respective DMSO-treated mESCs). Non-treated (serum) cells are included. Scale bar: 400μm. (C) Quantitative representation of number of BrdU positive (BrdU+) mESCs treated with indicated BIO concentrations or DMSO for 72h, as control (n = 3). (D) Cell cycle FACS analysis after propidium iodide staining of mESCs treated with BIO or DMSO for 72h (n = 3; BIO-treated compared to respective DMSO-treated mESCs). (E) Cycling index (S+G2M/G0G1) of experiments shown in D (n = 3). (F) mESCs were modified to express the FUCCI G1 phase reporter. Cells were treated for 72h either with PBS, 300ng/ml or 600ng/ml purified Wnt3a or alternatively with DMSO (0.06%) or 3 and 6 μM of CHIR99021 and analysed by FACS (Three technical replicates). All pooled data are represented as means ± SD. The asterisks indicate statistical significance by two-tailed Student’s t-test analysis (* p<0.05; ** p<0.01; ***p<0.001).