Tcf4 t5817

Wild-type STAT3 was purchased from Genediscovery Biotechnology. The tyrosine mutants (STAT3^Y705F and STAT3^S727A) were constructed by site-directed mutagenesis using the wild-type STAT3 cDNA template [33 (link)]. The correct sequence of the clones was verified by sequencing. Antibodies against the following proteins were used: CD38 (555459), CD44 (555478), CD45 (555485), and E-cadherin (610181) (all from BD Biosciences); CD105 (130-098-774) and CD133 (130-080-801) (both from Miltenyi Biotec); CD83 (11-0839-42; eBioscience); Vimentin (IF01; Calbiochem); CD151 (FAB1884P; R&D Systems); IL6R (sc-373708) and STAT3 (sc-8019) (both from Santa Cruz Biotechnology); GATA3 (ab199428), LIFR (ab202847), and Twist1 (Ab50887) (all from Abcam); β-actin (A5441), β-catenin (C2206) and TCF4 (T5817) (all from Sigma); and ERK (9102S), Snail1 (3895S), phospho-ERK (9101S; phosphorylated at T202/Y204), phospho-STAT3 (9138; phosphorylated at Y705) and phospho-STAT3 (9136; phosphorylated at S727) (all from Cell Signaling Technology).

The E-cadherin–GFP expression construct was a gift from Alpha Yap^{50 (link)} (University of Queensland, Queensland, Australia). Wild-type E-cadherin was generated by PCR amplification of the corresponding cDNA fragments using E-cadherin–GFP as a template. The E-cadherin tyrosine-to-alanine (⁷⁵³YYY^755→753AAA⁷⁵⁵) mutant was generated by site-directed mutagenesis using wild-type E-cadherin as a template. The correct clone sequence was verified by sequencing. Lentiviral-TOP-dGFP-reporter plasmids were obtained from Addgene (Addgene plasmid 14715, Cambridge, MA, USA). A pLKO.1-shRNA encoding an shRNA with a scrambled sequence or sequences targeting human Sox15 and E-cadherin, purchased from the National RNAi Core Facility, Taiwan, was introduced into HEK293T cells using the lentiviral packaging vectors pMD.G and pCMV_8.91. Antibodies against the following proteins were used: E-cadherin (610181; BD Biosciences, San Jose, CA, USA); histone H3 (Ab1791) and ABCG2 (Ab3380) (all from Abcam, Cambridge, MA, USA); active β-catenin (ALX-804-260/1; dephosphorylated at Ser33/37; Enzo Life Sciences, Farmingdale, NY, USA); total β-catenin (C2206), β-actin (A5441) and TCF4 (T5817) (all from Sigma-Aldrich, St Louis, MO, USA); CD133 (3663) and phosphorylated β-catenin (phosphorylated at Ser33/37/Thr41) (9561) (all from Cell Signaling Technology, Beverly, MA, USA).