Journal: BMC Biology
Article Title: A ZEB1-Neon knock-in uncovers traceable dynamics of epithelial-mesenchymal transition in tumors in vivo
doi: 10.1186/s12915-026-02629-0
Figure Lengend Snippet: Endogenous tagging of ZEB1 by mNeonGreen (Neon) with CRISPaint faithfully reports on ZEB1 expression in MDA-MB-231 and MCF10A cells. A Schematic representation of the CRISPaint approach, using a modified donor plasmid for seamless fusion at ZEB1 exon 9, integrating Neon-T2A-puro. sgRNA-directed expected Cas9 cuts in the ZEB1 locus and plasmid are indicated by red arrowheads. B Western blot analysis of successfully targeted MDA-MB-231 after clonal expansion of ZEB1-Neon clones E2, E9, E10, F11, A5, and B6 together with the parental (par) and one control clone (ctrl). Anti-ZEB1 immunoblotting identifies both ZEB1 wildtype and ZEB1-Neon proteins as indicated. C Immunofluorescence staining (IF) using anti-mNeonGreen and anti-ZEB1 antibodies of selected clones from B . Nuclei are stained with DAPI; arrowheads, ZEB1hi cells; open arrow, ZEB1lo cells. D Representative FACS plots and mean fluorescence intensity (MFI) of n = 4 experiments for MDA-MB-231 ZEB1-Neon, par and ctrl clones as in B . E Detection of intrinsic ZEB1-Neon fluorescence and DIC (BF) imaging using spinning-disc confocal imaging of clones from B . F , G Western blot of MCF10A par, two ctrl (E9, E11), and two successfully CRISPaint-targeted clones (B6, D9) in standard culture ( F ) and upon TGFβ treatment for 14 days of par, F8 ctrl, and D9 Zeb1-Neon clones ( G ). For anti-Neon detection in G , samples were run on opposing ends of the same membrane and merged (vertical separation). H IF imaging of F8 ctrl and D9 MCF10A clones with and without TGFβ treatment for 14 days as in C ; arrowheads, ZEB1hi cells; open arrow, ZEB1lo cells. Scale bars, 50 µm ( C , H ) and 20 µm ( E )
Article Snippet: MDA-MB-231 and MCF10A were purchased from American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 31966021)/10% FBS (Gibco, 10500064) and DMEM/F-12 (Gibco, 31331028)/5% horse serum (Gibco, 16050122)/20 ng/ml EGF (Peprotech, 100–15)/0.5 mg/ml hydrocortisone (Sigma, H0888)/0.1 mg/ml cholera toxin (Sigma, C8052)/10 mg/ml insulin (I9278), respectively, at 37 °C/5% CO 2 in a humidified incubator as described previously [ ].
Techniques: Expressing, Modification, Plasmid Preparation, Western Blot, Clone Assay, Control, Immunofluorescence, Staining, Fluorescence, Imaging, Membrane
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