Figures and data
Mutated β-catenin regulates exosome biogenesis gene expression in HepG2 cells.
(a-f) HepG2 cells were transfected with siRNAs targeting mutated β- catenin or control siRNAs. (a) Volcano plot of deregulated genes identified by microarray based-transcriptomic analysis. Red and blue dots indicated respectively significantly up- and down-regulated genes. (b) Upregulation of cellular component genes using FunRich software. (c) Upregulated genes associated with exosome biogenesis pathway. The graph indicates the fold change (FC) when comparing the mutated β-catenin silencing condition with the control condition. (d) Volcano plot of deregulated proteins identified by mass spectrometry. Red and blue dots indicated respectively significantly up- and down-regulated proteins. (e) Upregulation of cellular component proteins identified using FunRich software. (f) Upregulated proteins associated with exosome biogenesis pathway. The graph indicates the fold change when comparing the mutated β-catenin silencing condition with the control condition. Results are expressed as Mean ± SEM, two-tailed Student’s t-test analysis. *P<0.05;**P<0.01; ***P<0.001; ****P<0.0001.
Mutated β-catenin controls exosome secretion in HepG2 cells.
(a-g) HepG2-shβCat MUT and HepG2-shCtrl cells were treated with doxycycline (DOX) to silence or not mutated β-catenin. (a) Analysis of β-catenin and CyclinD1 expression by western-blot. Stain free was used as loading control. Graphs show the quantification of seven independent experiments. (b) Nanoparticle tracking analysis of supernatant. Graphs show the quantification of seven independent experiments. (c) Quantification of CD63-pHluorin MVB–PM fusion events visualized by live TIRF microscopy. Depicted data are representative of three independent experiments, each dot represents one cell. Images represent the cell mask (white) and red dots corresponding to fusion events. Scale bar: 10µm. (d) Extracellular vesicles (EVs) isolation protocol. Created with BioRender.com. (e) Analysis of β-catenin and CD63 expression in HepG2-derived EVs. Stain free was used as loading control. Graphs show the quantification of four independent experiments. (f) Transmission electron microscopy images of HepG2-derived EVs by close-up. Scale bar: 100nm. The graph shows the diameter quantification of EVs (n=93). (g) Electron microscopy images of HepG2 shCtrl and shβcat MUT cells showing multivesicular bodies (MVBs) (yellow arrowheads). Scale bar: 2 µm (zoom: 500 nm). The graphs show the quantification of the number of MVBs per cell and the MVB diameter. (a-f) Results are expressed as Mean ± SEM, two-tailed Student’s t-test analysis. *P<0.05; **P<0.01; ***P<0.001; ns, non-significant.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Activated β-catenin represses SDC4 and RAB27A expression in liver cancer cells.
(a-d) HepG2 cells were treated with doxycycline (DOX) to express either a control shRNA (shCtrl) or a shRNA targeting mutated β-catenin (shβcat MUT). (a) Analysis of SDC4 and RAB27A mRNA expression by qRT-PCR. Graphs show the quantification. (b) Analysis of Rab27a protein expression by western-blot. Stain free was used as loading control. The graph shows the quantification. (c-d) Epifluorescence images of HepG2 shCtrl and shβcat MUT cells stained with Syndecan-4 and Rab27a antibodies (red), Phalloidin (green), Hoechst (blue). Scale bar: 20 µm. Graphs show the quantification of the fluorescence intensity per image and divided per nuclei. Depicted data are representative of three independent experiments, each dot represents one image. (e) Analysis of SDC4 and RAB27A mRNA expression in Huh6 cells expressing either a control shRNA (shCtrl) or a shRNA targeting β-catenin (shβcat) treated with DOX. The graph shows the quantification. (f) Analysis of SDC4 and RAB27A mRNA expression in SNU398 cells expressing either a control shRNA (shCtrl) or a shRNA targeting β-catenin (shβcat) treated with DOX. The graph shows the quantification. (g-h) Analysis of Rab27a protein expression in Huh6 (g) or SNU398 (h) cells expressing either a control shRNA (shCtrl) or a shRNA targeting β-catenin (shβcat). Stain free was used as loading control. The graphs show the quantification. (i-m) Huh7 cells treated with DMSO or CHIR99021 (3µM) for 48 hours. (i) Analysis of β-catenin expression by western-blot. The graph shows the quantification. (j) Epifluorescence images of cells stained with β-catenin antibody (red), Phalloidin (green), Hoechst (blue). Scale bar: 20 µm. (k) Analysis of SDC4 and RAB27A mRNA expression by qRT-PCR. The graphs show the quantification. (l) Analysis of Rab27a protein expression by western-blot. The graph shows the quantification. (m) Quantification of CD63- pHluorin MVB–PM fusion events visualized by live TIRF microscopy. Depicted data are representative of three independent experiments; each dot represents one cell. Images represent the cell mask (white) and red dots corresponding to fusion events. (a-m) All graphs show the quantification of at least three independent experiments. Results are expressed as Mean ± SEM, two-tailed Student’s t-test analysis. *P<0.05; **P<0.01; ***P<0.001.
Activated β-catenin represses immune infiltration in liver cancer spheroids through exosomes. (a)
Peripheral Blood Mononuclear Cells (PBMC) infiltration analysis protocol. Created with BioRender.com. (b) Images of HepG2 spheroids expressing a control shRNA incubated or not with PBMC for 24 hours. (c) Analysis of PBMC infiltration and tumor cell survival in Huh7 spheroids treated with DMSO or CHIR99021. (d) Analysis of PBMC infiltration and tumor cell survival in HepG2 spheroids expressing control shRNA (shCtrl) or shRNA targeting mutated β- catenin (shβcat MUT). (e) Analysis of PBMC infiltration and tumor cell survival in HepG2 spheroids co-expressing shRNA targeting mutated β-catenin (shβcat MUT) and control siRNA (siCtrl) or siRNA targeting Rab27A (siRab27A). (c-e) Graphs show the quantification of six independent experiments. Results are expressed as Mean ± SEM, one or two-tailed Student’s t-test analysis. *P<0.05; **P<0.01; ***P<0.001.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Downregulation of SDC4 and RAB27A in human HCC samples with CTNNB1 mutations. (a)
Analysis of SDC4 and RAB27A mRNA expression in β- catenin mutated (red) and non-mutated (blue) HCC (Cbioportal cohort, n=366). (b) Analysis of SDC4 and RAB27A mRNA expression in β-catenin mutated (red) and non-mutated (blue) HCC (Boyault et al. cohort, n=56). (c) Immunohistochemistry (IHC) analysis of glutamine synthetase (GS) and Rab27a in HCC samples presenting or not β-catenin mutations. Scale bar: 1mm. (T: tumoral, NT: non tumoral). The upper graphs show the quantification of the percentage of cells positive for GS or Rab27A. The analysis was split into two cohorts (circle and triangle). The lower graph shows the Pearson correlation between Rab27a and GS protein expression (n=56). Results are expressed as Mean ± SEM, two-tailed Student’s t-test analysis or Pearson correlation test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
