human colon cancer cell line hct116  (ATCC)

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: (A) Schematic workflow of the self-renewal model. Hakai-silencing was induced in HT29 monolayer cultures and after 72 hours, cells were cultured in cancer stem cell promoting conditions for 5 days to form tumourspheres. (B) Tumourspheres were phenotypical characterized by phase contrast images. Representative images of Hakai-silenced HT29 tumourspheres and control conditions were taken after 5 days of the induction of stemness. Images were obtained using a 10x objective. Scale bar, 200 µm. (C) Quantification of the number of HT29 tumourspheres in Hakai-silenced compared to control conditions. (D) Size of tumourspheres of HT29 tumourspheres in Hakai-silenced compared to control conditions. Results are expressed as mean ± SEM. Quantification was carried out using ImageJ software, and statistical significance was determined using GraphPad Prism software. (E) mRNA expression levels of Hakai (CBLL1 gene) and stem cell markers in HT29 tumourspheres in Hakai-silenced compared to control analysed by RT-PCR. (F) Expression of Hakai and stem cell protein markers was determined by Western blot in HT29 tumourspheres with Hakai silencing compared to control. Protein bands were quantified using ImageJ and normalized to loading control. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using an unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Cell Culture, Control, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: (A) Schematic workflow of the proteomic study in Hakai-silenced tumoursphere compared to control. Hakai-silencing in HT29 cells was induced and cells were seeded in stem cell promoting media using ultra-low attachment plates to induce tumoursphere formation. Protein extracts were digested and protein profile was analysed by silver staining in SDS-page gels. Proteins were digested with trypsin and the obtained peptides were fractionated and separated by nano-LC-MS/MS analysis coupled to a timsTOF Pro mass spectrometer. (B) Hakai-silencing in tumourspheres was confirmed in three biological replicates by Western blot. GAPDH was used as loading control. (C) Venn diagram of identified proteins (≥ 2 peptides) in tumourspheres obtained from Hakai-silenced HT29 colon cancer tumourspheres versus control. (D) Principle Component Analysis (PCA) plot showing two principal components corresponding to three replicates of Hakai-silenced tumourspheres and three replicates of control tumourspheres. (E) Heatmap showing the regulated proteins. Up and down regulated proteins in Hakai-silenced tumoursphere versus control are represented. (F) Volcano plot of differentially expressed proteins in Hakai-silenced tumourspheres versus control tumourspheres. Black dots represent proteins showing significant fold changes. The protein significance was set to adjusted p-value < 0.05, protein fold change to ≥ 1.5, used peptides to ≥ 2. (G) Box plot showing the protein profiling comparison, based on the label-free quantitation (LFQ) for which levels changed significantly between samples. The LFQ values are plotted on a Log2 scale along the vertical axis. (H) Top 20 Hakai (CBLL1 gene) substrates predicted in Homo sapiens (blue) and the 4 known substrates already described (red) by using UbiBrowser 2.0 database.

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Control, Silver Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Comparison, Quantitation Assay

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet:

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Multiplex sample analysis, Control, Significance Assay

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: (A) Hakai overexpression reduces LRP4 protein levels in HEK293 and HCT116 cells shown by Western blot. (B) Impact of Hakai on cell-surface expression of LRP4 in HEK293T transfected with LRP4 and empty vector or Hakai-V5. Surface levels of LRP4 were detected in unpermeabilized cells after which total levels of Hakai were visualized via permeabilization using confocal microscopy. Pictures were taken using a 63x objective and scale bar represents 30 µm. (C) Coimmunoprecipitation of Hakai and LRP4 in HEK293 cells overexpressing FLAG-Hakai, LRP4 and HA-ubiquitin. (D) Hakai-dependent ubiquitination of LRP4. FLAG-Hakai, HA-ubiquitin and LRP4 were transiently transfected into HEK293 cells. Immunoprecipitation was performed with anti-LRP4 and analyzed by Western blot. (E) LRP4 and FLAG-Hakai levels in HCT116 cells treated with the proteasome inhibitor MG132 analysed by Western blot. β-catenin were used as a positive control for MG132 treatment. (F) HCT116 cells were transiently transfected with FLAG-Hakai and the next day treated with MG132 (30 µM) for 6 h. Endogenous LRP4 levels were analysed by Western blot. (G) HCT116 cells were transiently transfected with FLAG-Hakai and treated with 50 µM of Hakin-1. LRP4 endogenous levels were analysed by Western Blot.

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Ubiquitin Proteomics, Immunoprecipitation, Positive Control

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: Endogenous LRP4 and Hakai levels in HCT116 cells treated with (A) lysosome inhibitor Chloroquine and (B) autophagy inhibitor 3-MA analyzed by Western blot. LC3 I/II was used as a positive control for chloroquine and 3-MA treatment and GAPDH as a loading control.

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Western Blot, Positive Control, Control

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: Wnt signalling activity upon overexpression of the indicated plasmid in colon cancer HCT116 treated with control L-cell conditioned medium (LCM) and WNT3A conditioned medium (WCM). Cells were transfected with TOP-flash or FOP-flash reporter plasmids together with the indicated plasmids (A) Hakai-V5 and/or LRP4; (B) Hakai-V5 and/or LGR5 and (C) Hakai-V5, LRP4 and/or LRP6. Relative luciferase activity is presented as fold of control in mean ± SEM of three independent experiments.Two-ways ANOVA with Dunnet’s post hoc analysis was performed for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, Control, Transfection, Luciferase

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: (A) Schematic representation of the experimental workflow used to assess self-renewal. Hakin-1 (50 µM) was added at the initiation of cancer stem cell induction. (B) Representative images of HT29 tumourspheres treated with Hakin-1 or DMSO (vehicle control) five days after induction under stemness-promoting conditions. Images were acquired using a using a 4x (upper images, scale bar: 250 µm) or 10x (lower images, scale bar: 200 µm) objective. (C) Quantification of the number of tumourspheres formed from HT29 cells treated with Hakin-1 or DMSO. (D) Quantification of tumoursphere size in the same conditions. Data in (C) and (D) are presented as mean ± SEM. Quantification was performed using ImageJ software, and statistical analysis was conducted with GraphPad Prism. (E-F) Effect of Hakin-1 on Wnt/β-catenin signalling and stem cell markers at mRNA levels. RT-qPCR analysis of the expression of LEF-1 and TCF-1 transcription factors, stem cell markers LGR5 and NANOG (E), and CCND1, MMP7 and C-MYC (F). (G) Protein expression levels of E-cadherin (differentiation marker), LGR5, Hakai and NANOG (stem cell markers) upon treatment with Hakin-1 (50 µM) were analysed by Western blot, using the indicated antibodies. Vinculin was used as loading control. Protein bands were quantified using ImageJ and normalized to loading control. Results are expressed as mean ± SEM. T-test was performed for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Control, Software, Quantitative RT-PCR, Expressing, Marker, Western Blot

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: (A) Schematic workflow. Tumourspheres already formed were treated with Hakin-1. (B) Representative images of tumourspheres in HT29 treated with DMSO (control) or Hakain-1 in day 8 after stemness condition a 4x (upper images, scale bar: 250 μm) or 10x (lower images, scale bar: 200 μm) objective. (C) Size of tumourspheres derived from HT29 cells treated with DMSO (control) or 50 μM Hakin-1. Surface area quantification was performed using ImageJ software. Tumoursphere area of at least 15 tumourspheres per experiment were measured. (D) Quantification of the number of tumourspheres after DMSO (control) or Hakin-1 treatment by self-renewal assay. Results are expressed as mean ± SEM and statistical analysis was performed using a t-test of GraphPad Prism software (***p < 0.001 and ****p < 0.0001).

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Control, Derivative Assay, Software

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: Immunofluorescence analysis of HT29 tumourspheres treated with DMSO (vehicle control) or Hakin-1 (50 µM). (A–D) Left panels: representative immunofluorescence images of E-cadherin (A), LGR5 (B), LRP4 (C), and MUC2 (D). Images were captured using a 20x objective (scale bar = 50 µm); magnified images (circle images) were generated by digital zoom. Right panels: quantitative analysis of marker expression. For E-cadherin, LRP4, and LGR5, fluorescence intensity was normalized to area and quantified across at least 5 tumourspheres per condition, shown as scatter plots. For MUC2, the number of positive cells was quantified by measuring the stained area and normalizing to DAPI-stained nuclear area. Data are presented as mean ± SEM from three independent experiments. Statistical significance was calculated using unpaired t -tests (*p < 0.05, **p < 0.01, ***p < 0.001) in GraphPad Prism.

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Immunofluorescence, Control, Generated, Marker, Expressing, Fluorescence, Staining

Journal: bioRxiv

Article Title: E3 ubiquitin-ligase Hakai induces LRP4 degradation and regulates Wnt/β-catenin signalling in colorectal cancer cells

doi: 10.1101/2025.11.20.689197

Figure Lengend Snippet: Levels of Hakai and PRPS2 proteins in Hakai-silenced HT29 colon cancer tumourspheres versus control tumoursphere were assessed by Western blot. GAPDH was used as loading control.

Article Snippet: Human colon cancer cell lines HCT116 (ATCC®#CCL-247TM) were obtained from the ATCC and were grown in DMEM.

Techniques: Control, Western Blot