Article Title: An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance
Figure Lengend Snippet: SCF FBXW7 interacts and targets ZEB2 for degradation in a GSK-3β phosphorylation-dependent manner. a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week fbxw 7 fl/fl and fbxw 7 ΔG mice. Yellow circles in the lower panel denote potential Fbxw7-associated proteins. a Right, WB analysis (upper panels), and RT-PCR analysis (lower panels) of fbxw 7 fl/fl vs. fbxw 7 ΔG derived crypts and intestinal proteins and mRNA expression for ZEB2 and β-actin control. Experiments were performed on at least three independent occasions. b Left, schematic representation of the modified yeast two-hybrid reverse Ras Recruitment Screening (rRRS) system identifying proteins interacting with Fbxw7 in a GSK-3β phosphorylation-dependent manner. GSK-3β under the control of the methionine-regulated MET 3 promoter induces phosphorylation of encoded myristoylated proteins through a cDNA library plus positive control expressing FLAG-β-catenin (B—Middle) which only rescued the growth of cdc25–2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7ΔF (i.e. human FBXW7α isoform mutant lacking F-box domain; therefore, interaction with Skp1 is lost and degradation of SCF Fbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37 °C, in a methionine-dependent manner. In the FBXW7ΔF mutant, both the N-terminal F-box and Dim-domains are deleted to avoid any interactions with SKP1 and other FBXW7 isoform-associated proteins. Thus, cdc25–2 mutant yeasts can grow only at 37 °C, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7ΔF takes place. The FBXW7ΔF(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37 °C (B—Right). Red circles show the GSK-3β-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B—Right). c Left, subcellular localisation of GFP-fused human ZEB2 in the absence (top; nuclear) and presence (bottom; nuclear spots indicative of protein degradation) of GSK-3β in HCT116 CRC cells. ( c— Middle and c— Right) WB analysis of total ZEB2 protein level following the inhibition of GSK-3β (e.g. WS119 or LiCl treatment, and siRNA against GSK-3β) and of UPS pathways (MG132) in SW620 CRC cells. d Direct binding and ubiquitin-dependent degradation of ZEB2 by FBXW7. Co-immunoprecipitation (IP) of ZEB2 upon pull-down of FBXW7 in HEK-293T cells (Left); co-IP of FBXW7 upon pull-down of ZEB2 using the TNT-coupled reticulocyte lysate (Middle), and ubiquitination assays with HA-tagged ubiquitin- (HA-Ub) expressing construct in HEK-293T cells (Right). The asterisk indicates a nonspecific band(s). e Co-IP of endogenous ZEB2 upon pull-down of FBXW7 in HCT116 cells with FBXW 7 deletion. f ZEB2 pulse-chase stability assays with 15 µg/ml cycloheximide (CHX) in HCT116 cells with or without FBXW 7 deletion
Article Snippet: We then conducted a similar cDNA array, where fbxw 7ΔG organoids cocultured with IMFfl/fl and IMFΔG fibroblasts, respectively, at day 1 (EpΔG IMFfl/fl -derived organoids vs. EpΔG IMF∆G -derived organoids).
Techniques: Two-Dimensional Gel Electrophoresis, Mass Spectrometry, Isolation, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Modification, cDNA Library Assay, Positive Control, Mutagenesis, Clone Assay, Inhibition, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Construct, Pulse Chase