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Fig. 2 | Molecular Cancer

Fig. 2

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

Fig. 2

shFBXO11 preferentially attenuates tumor initiation of non-EMT-like cells. a Non-EMT-like cells (left) and EMT-like cells (right) transduced with SCR or shFBXO11 and inoculated in NOG mice (103 cells per injection, n = 6 to 10 injections/group). Western blots (first columns of each panel) illustrate FBXO11 and β-actin expression in inoculated cells. Representative bioluminescent (BL) signal of mice shows a delay in tumor initiation only in mice inoculated with shFBXO11 non-EMT-like cells. b Relative quantification of tumor growth illustrated in (a). BL signals of each clone are measured as total flux and normalized by total flux of its corresponding SCR-transduced clone at week 6. Tumor growth is significantly inhibited only in the FBXO11-depleted non-EMT-like cells (asterisk indicates p < 0.05 tested by ANOVA with Tukey’s test). Error bars represent SEM. c Sanger sequencing confirming three different FBXO11 indel clones (D7, F4 and G11) as compared to sgRNA target sequence of the CRISPR-Cas9 gene-editing system. Intact sequence is shown in non-EMT cells and inserted nucleotides are marked in red. F4 clone gained the same indel mutations in both alleles. d Western blot shows that FBXO11 indel clones do not express FBXO11 proteins. e Tumor volume of non-EMT and FBXO11 indel clone xenografts as measured in week 6 after injection of 103 cells (n = 6 injections/group, asterisks indicate p < 0.00005 by ANOVA with Tukey’s test). Error bars represent SD. f Western blot of whole lysates isolated from non-EMT-like- and EMT-like cells transduced with SCR or shFBXO11 and stained for FBXO11 and p21 shows that p21 is exclusively induced by shFBOX11 in non-EMT-like cells (upper). Western blot of whole lysates of non-EMT-like cells transduced with either or both of shFBXO11 and shp53 and stained for p53, p21, and β-actin. shFBXO11-mediated induction of p21 relies on p53 protein (lower). g FBXO11 indel clones significantly induce p21 protein. The percentage of immunostained p21+ cells in a total of approximately 1000 cells, was automatically counted with image J in triplicates (asterisks indicate p < 0.0005 by ANOVA with Tukey’s test). Error bars represent SD. h Quantification of cell proliferation of GFP+ cells as influenced by shFBXO11 and shp53 shows that the shFBXO11-induced growth reduction can be partly rescued by shp53 (asterisks ** and * indicate p < 0.0001 and p < 0.005 by t-test, respectively). Error bars represent SD. i Quantification of cell number upon FBXO11 knockout in non-EMT-like cells shows that cell number in culture is significantly reduced by FBXO11 knockout. Equal numbers (105) of two different FBXO11 indel clones (G11 and F4) or the control cells (non-EMT-like) were cultured for 8 days prior to cell-counting (asterisk indicates p < 0.01 tested by ANOVA with Tukey’s test). Error bars, SD. j Quantification of invasive capacity of 5 × 104 control non-EMT-like cells or a FBXO11 indel clone, F4 on Matrigel-coated filters in 72 h. Note that invasion is significantly abolished by deletion of FBXO11 (asterisk indicates p < 0.05 by t-test). Error bars represent SD of averages of quadruplicates in two independent experiments. k Depiction of the model used for testing in vitro collective migration (top) and cell invasion (bottom right). The clusters of mixture of GFP-labeled and unlabeled non-EMT-like cells (1:1 ratio) were either plated in an adhesion culture for 24 h (hrs) (bottom left) or plated in an invasion assay incubated for 72 h (bottom right), followed by staining for K19 (red) and GFP (green). Dual fluorescence imaging allows for the demonstration of doublets of green/red cells migrating through individual pores by collective migration. Scale bars, 50 μm

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