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