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Figure 1 | BMC Cancer

Figure 1

From: E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition

Figure 1

Characterization of MCF10A CDH1-/- cells. a) CDH1 sequence from MCF10A CDH1-/- and wildtype MCF10A cell lines depicting the engineered 4 bp deletions as determined by RNAseq. The specified deletion was attributed to ZFN editing on exon 11 of both CDH1 alleles in MCF10A CDH1-/- cells. The ZFN binding site is represented by bases in red uppercase and the ZFN cut site is represented in red lowercase. b) Immunoblot of MCF10A CDH1-/- confirming the loss of E-cadherin expression as a result of the 4 bp deletion with α-actin as loading control. The cropped images are a composite of the same nitrocellulose immunobloted with antibodies against E-cadherin followed by α-actin (Additional file 1: Figure S1). c) Immunofluorescence showing loss of E-cadherin from the cell junctions in MCF10A CDH1-/- but not wildtype MCF10A cells. d) Comparison of growth morphology between MCF10A CDH1-/- and wildtype at subconfluence and full confluence. At subconfluence, MCF10A CDH1-/- showed clustered and contracted distribution while some wiltdtype MCF10A cells exhibited more mesenchymal morphology. At full confluence, both isogenic cells retained epithelial cobblestone-like morphology, although MCF10A CDH1-/- displayed gaps not observed in wildtype cells. e) Comparing cell proliferation profile between both MCF10A isogenic cells. A measure of cell proliferation was represented by the normalized cell index taken from impedence measurements generated by cells grown over 96 h on a 96-well E-Plate on the xCELLigence. f) The time course of cell migration was quantified using IncuCyte wound confluence at 1 h intervals over 35 h. MCF10A CDH1-/- cells were shown to take significantly longer in wound closing compared to wildtype cells. g) Soft agar assay to determine anchorage-independent growth as a result of E-cadherin loss. Anchorage-independent growth was observed only in the positive control MCF-7 cells, but not in either of the MCF10A isogenic cells. Representative images from one of two biological replicates were presented.

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