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Fig. 1 | Molecular Cancer

Fig. 1

From: Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity

Fig. 1

Characterization of MCF7 breast cancer cell clones as a model of tumor aggressiveness and FBXO11 as a functional readout. a RT-qPCR analysis of relative EMT marker expression shows upregulation of epithelial markers CDH1 and OCLN in non-EMT cells and upregulation of mesenchymal markers SNAI2 and TWIST in EMT-like cells. E-cadherin is encoded by CDH1; occludin by OCLN. Error bars represent SD of quadruplicates. b Monolayer culture non-EMT-like (left column) and EMT-like (right column) MCF7 clones stained (green) with ZO-1 (upper row), Occludin (middle row), E-cadherin (lower row), and DAPI (blue, nuclei). Only the non-EMT-like clone exhibits distinct staining at the cell-cell junctions. Bar = 50 μm. c Representative BLI of NOG mice inoculated with 104 non-EMT-like (left column) or EMT-like (right column) MCF7-pFU-L2G cells (n = 6 inoculations/cell-type). Time indicates weeks after inoculation. Non-EMT-like cells grow to a larger size than the EMT-like cells. d Quantification of tumor sizes measured as total flux by in vivo BLI at week 11 (n = 6 injections/group) (asterisk indicates p < 0.005 tested by t-test). Error bars represent SEM. e Representative immunofluorescence staining of human-specific K19 (red) on mouse lung sections reveals non-EMT-like-derived metastases (n = 4 mice/group). Metastatic colonies stained with K19 are only found in lungs of mice injected with non-EMT-like cells. Nuclei (blue) and scale bar, 50 μm. f Relative shRNA distributions in Non-EMT-like (left) and EMT-like (right) MCF7 cells 20 days after transduction with a lentiviral human-specific epigenetic shRNA library (referred as epi-library) in ascending order. The result is representative of 2 replicates. Negative controls, SCRs, are indicated by green and shFBXO11s in red. shFBXO11s are the most depleted shRNAs in the non-EMT-like cells compared to the EMT-like cells. g Kaplan-Meier plots of RFS (top) and OS (bottom) of breast cancer patients stratified by mRNA expression of FBXO11 using a web based survival analysis tool (kmplot.com). A total of 30 cohorts (3951 patients) are split into low (n = 1861) vs. high (n = 2090) expression groups for RFS (Hazard ratio (HR) = 1.46 (1.31–1.63), log rank p = 2 × 10− 11), and 11 cohorts (1402 patients) are split into low (n = 468) vs. high (n = 934) expression groups for OS (HR = 1.37 (1.08–1.74), log rank p = 0.01). h FBXO11 stains strongly in highly differentiated breast carcinomas. Representative immunostaining (brown) of MUC1 (top) or FBXO11 (bottom) out of six highly differentiated luminal breast carcinomas. Biopsy 1 is correctly (left panel) and biopsy 2 is inversely polarized (right panel) based on MUC1 expression and both express FBXO11 in the nuclei. Nuclei (blue) and scale bar, 50 μm

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