Fig 1: IGF2BP1 sustains mesenchymal-like tumor cell properties. (A) Indicated tumor-derived cell lines were cultured (48 h) and harvested at 80% confluence before analyzing the abundance of indicated proteins by western blotting. Epithelial-like cell lines and marker proteins are labeled in green. Mesenchymal-like cell lines and marker proteins are depicted in red. VCL and HSPB1 (HSP27) served as loading controls. IGF2BP1 is almost exclusively expressed in the following tumor-derived mesenchymal-like cells: ES-2 ovarian carcinoma (ATCC#: CRL-1978); SW480 colorectal carcinoma (ATCC#: CCL228); MDA-MB-231 (ATCC#: HTB-26) and HBL-100 (ATCC#: HTB-124) mammary carcinoma; 1F6 (no ATCC# available) and HT-144 (ATCC#: HTB-63) melanoma. In epithelial-like adenocarcinoma-derived cells [OVCAR (ATCC#: HTB-161) ovarian adenocarcinoma; MCF7 (ATCC#: HTB-22) breast adenocarcinoma; HT-29 (ATCC#: HTB-38) colorectal adenocarcinoma], expression of IGF2BP1 was only observed in OVCAR cells. (B) Melanoma-derived HT-144 cells were transiently transfected with indicated siRNAs for 72 h. The abundance of indicated proteins was analyzed by western blotting. IGF2BP1 as well as LEF1 depletion result in reduced FN1 and SNAI2 protein abundance, whereas CDH1 and VIM levels remain essentially unchanged. (C–F) HT-144 cells were stably transduced by lentiviral vectors encoding IGF2BP1 (shI1-1), LEF1 (shL1-1), SNAI2 (shS2-1) directed or control (shC) shRNAs. Three weeks after transduction, cells were cultured for 48 h before analyzing protein abundance by western blotting with indicated antibodies (C and D). Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) using VCL and HSPB1 for cross-normalization, as indicated above panels (C). Standard deviation of at least three independent analyses is shown. The stable knockdown of SNAI2, LEF1 and IGF2BP1 promotes the expression of the epithelial marker CDH1, whereas all mesenchymal marker proteins were reduced. Cell morphology was monitored by bright field microscopy (E). Cells were cultured on collagen coated coverslips for 48 h before immunostaining of CTNNB1 and CTNND1 (p120 Catenin) to label cell–cell contacts (F). Enlargements of boxed regions (left panels) are shown in right panels (enlargement). All three stable knockdowns promote the formation of cell–cell contacts, suggesting an enhancement of epithelial-like cell morphology. (G and H) MCF7 cells were stably transduced with GFP-ZBP1 (the chicken ortholog of IGF2BP1) or GFP. Three weeks after transduction, cells were cultured for 48 h before determining the abundance of indicated proteins by western blotting (G). Cell morphology was monitored by bright field microscopy (H, left panel) and immunostaining for CDH1 as well as labeling of F-actin by phalloidin (H, right panel). Neither CDH1 expression nor cell-cell contact formation is compromised by GFP-ZBP1, although cell size appeared modestly increased. Bars, 10 µm.