Overexpression of β-catenin maintains B6 ESC self-renewal in the presence of LIF.

<p>(A) Immunoblot analysis of phospho-β-catenin, β-catenin and β-actin levels in B6 ES cells cultured in 1000 U/ml LIF condition supplemented without or with 3 µM CH for 4 days. (B) Immunofluorescence analysis of 3 µM CH-treated and non-treated B6 ESC clones stained for β-catenin and Hoechst for nuclei in the presence of LIF for 4 days. Scale bars represent 100 µm. (C) Quantitative RT-PCR analysis of <i>Axin2</i>, <i>T</i> and <i>Cdx1</i> transcript levels in 3 µM CH-treated and control B6 ES cells. Error bars represent the SD of three biological replicates. (D) β-catenin S33A protein stably expressed in ES cells was detected by immunoblotting. (E) Phase contrast microscopy image and alkaline phosphatase staining of control and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Scale bars represent 100 µm. (F) Immunofluorescence staining for OCT4, NANOG, and KLF4 in mock and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Cell nuclei were stained with Hoechst. Scale bars represent 100 µm. (G) Quantitative RT-PCR analysis of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 transcript levels in mock and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Error bars represent the SD of three biological replicates. (H) β-catenin S33AΔC, β-catenin S33A and TCF4ΔN protein were detected by immunoblotting with anti-flag tag antibody from extracts of ES cells transfected with PiggyBac plasmids. Quantitative RT-PCR analysis of Axin2, T and Cdx1 transcript levels in β-catenin S33AΔC, β-catenin S33A, TCF4ΔN, and mock-transfected B6 ES cells. Error bars represent the SD of three biological replicates. (I) Alkaline phosphatase staining of mock-transfected, and β-cateninΔC over-expressing B6 ES cells cultured in LIF for 5 passages, and mock-transfected and TCF4ΔN-transfected B6 ESCs cultured in CHIR99021 plus LIF for 5 passages. Scale bars represent 100 µm.