gsk3β wt Search Results


hct116-gsk3β-ko  (Cellectis sa)
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Cellectis sa hct116-gsk3β-ko
A. Overview of experimental design . <t>HCT116</t> cells (with stabilizing β-catenin mutation) with wild-type <t>(HCT116-GSK3β-WT)</t> or knockoutGSK3β (HCT116-GSK3β-KO) were profiled using LC-MS/MS to identify associated protein networks. B. Volcano plot of protein expression from mass-spectrometry study . Plot indicates gene symbols for identified proteins with log2 fold-change greater than |2| and p-value < 0.05 C. Heat map of protein expression . Showing replicated wild-type and knockout samples showing those proteins with altered expression (with log2 fold-change greater than |2| and p-value < 0.05)
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A. Overview of experimental design . HCT116 cells (with stabilizing β-catenin mutation) with wild-type (HCT116-GSK3β-WT) or knockoutGSK3β (HCT116-GSK3β-KO) were profiled using LC-MS/MS to identify associated protein networks. B. Volcano plot of protein expression from mass-spectrometry study . Plot indicates gene symbols for identified proteins with log2 fold-change greater than |2| and p-value < 0.05 C. Heat map of protein expression . Showing replicated wild-type and knockout samples showing those proteins with altered expression (with log2 fold-change greater than |2| and p-value < 0.05)

Journal: bioRxiv

Article Title: Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

doi: 10.1101/2021.01.26.428222

Figure Lengend Snippet: A. Overview of experimental design . HCT116 cells (with stabilizing β-catenin mutation) with wild-type (HCT116-GSK3β-WT) or knockoutGSK3β (HCT116-GSK3β-KO) were profiled using LC-MS/MS to identify associated protein networks. B. Volcano plot of protein expression from mass-spectrometry study . Plot indicates gene symbols for identified proteins with log2 fold-change greater than |2| and p-value < 0.05 C. Heat map of protein expression . Showing replicated wild-type and knockout samples showing those proteins with altered expression (with log2 fold-change greater than |2| and p-value < 0.05)

Article Snippet: HCT116-GSK3β-KO and paired isogenic control cell line HCT116-GSK3β-WT were created by integrating a ssDNA oligonucleotide containing several stop codons at the GSK3 β locus with TALEN technology (Cellectis).

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Expressing, Mass Spectrometry, Knock-Out

A. Schematic of the canonical Wnt signalling pathway and summary of key proteins analysed in this study . Proteins shaded grey showed no significant difference between knockout and wild type cells. Those shaded green were more abundant in wild-type cells. Western blot analysis of canonical Wnt components and GSK3β protein expression characterization in cell models . 15 μg of total protein lysates from each cell-line were analyzed on a single phase 8% SDS gel and alpha-actin used as loading control. All were replicated in at least n=3 unless indicated otherwise. Quantifications (arbitrary intensity units) are mean values ± SEM (Mann-Whitney U test p-value indicated). ( B ) Protein expression of GSK3β and β-Catenin in HCT116 cells (independently sourced isolate), HCT116-GSK3β-KO cells and HCT116-GSK3β-WT cells. ( C ) β-Catenin, ( D ) TCF3 and TCF4, ( E ) Axin, ( F ) DVL2 and DVL3 (n=2) (G) TopFLASH reporter assay of canonical Wnt signalling activity in HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cells were transfected with TopFlash plasmids, TopFlash luciferase activity was assessed 48 hours post-transfection following passive lysis of cells. Luciferase signaling was normalized to Renilla transfection control. P value 0.064, n=3.

Journal: bioRxiv

Article Title: Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

doi: 10.1101/2021.01.26.428222

Figure Lengend Snippet: A. Schematic of the canonical Wnt signalling pathway and summary of key proteins analysed in this study . Proteins shaded grey showed no significant difference between knockout and wild type cells. Those shaded green were more abundant in wild-type cells. Western blot analysis of canonical Wnt components and GSK3β protein expression characterization in cell models . 15 μg of total protein lysates from each cell-line were analyzed on a single phase 8% SDS gel and alpha-actin used as loading control. All were replicated in at least n=3 unless indicated otherwise. Quantifications (arbitrary intensity units) are mean values ± SEM (Mann-Whitney U test p-value indicated). ( B ) Protein expression of GSK3β and β-Catenin in HCT116 cells (independently sourced isolate), HCT116-GSK3β-KO cells and HCT116-GSK3β-WT cells. ( C ) β-Catenin, ( D ) TCF3 and TCF4, ( E ) Axin, ( F ) DVL2 and DVL3 (n=2) (G) TopFLASH reporter assay of canonical Wnt signalling activity in HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cells were transfected with TopFlash plasmids, TopFlash luciferase activity was assessed 48 hours post-transfection following passive lysis of cells. Luciferase signaling was normalized to Renilla transfection control. P value 0.064, n=3.

Article Snippet: HCT116-GSK3β-KO and paired isogenic control cell line HCT116-GSK3β-WT were created by integrating a ssDNA oligonucleotide containing several stop codons at the GSK3 β locus with TALEN technology (Cellectis).

Techniques: Knock-Out, Western Blot, Expressing, SDS-Gel, MANN-WHITNEY, Reporter Assay, Activity Assay, Transfection, Luciferase, Lysis

A. Western blot analysis and mass spectrometry analysis of cell-cell adhesion proteins . 15 μg of total protein lysates from HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were analysed by western blot analysis for protein expression of GSK3β, PKP2, PKP3, and JUP. Protein abundance of Actin was used as a loading control. n = 3. Graphical representation of normalized fmol on column protein abundance from mass spectrometry analysis of HCT116-GSK3β-KO cells. * = P-value =<0.05.

Journal: bioRxiv

Article Title: Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

doi: 10.1101/2021.01.26.428222

Figure Lengend Snippet: A. Western blot analysis and mass spectrometry analysis of cell-cell adhesion proteins . 15 μg of total protein lysates from HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were analysed by western blot analysis for protein expression of GSK3β, PKP2, PKP3, and JUP. Protein abundance of Actin was used as a loading control. n = 3. Graphical representation of normalized fmol on column protein abundance from mass spectrometry analysis of HCT116-GSK3β-KO cells. * = P-value =<0.05.

Article Snippet: HCT116-GSK3β-KO and paired isogenic control cell line HCT116-GSK3β-WT were created by integrating a ssDNA oligonucleotide containing several stop codons at the GSK3 β locus with TALEN technology (Cellectis).

Techniques: Western Blot, Mass Spectrometry, Expressing

A. Mechanical stress test of cell-cell adhesion . Monolayer cultures of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were detached from culture surface and cell adhesions stressed with mechanical agitation. B. Cell migration assay . HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were grown until confluent and then cell cycle arrested with 24-hour treatment of 2mM thymidine. Migration scratch was then made in the monolayer and capacity of cells to migrate into the free space assessed by light microscopy. Movement of cell migration front was measured as area free of cells and percentage migration calculated and presented graphically. Graphical representation of cell migration is presented as percentage closure of scratch. C. Soft agar colony growth assay Cells were seeded individually in soft agar matrix supplemented with complete growth media and colony formation was assessed every 24 hours over a 6 day period by light microscopy.

Journal: bioRxiv

Article Title: Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

doi: 10.1101/2021.01.26.428222

Figure Lengend Snippet: A. Mechanical stress test of cell-cell adhesion . Monolayer cultures of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were detached from culture surface and cell adhesions stressed with mechanical agitation. B. Cell migration assay . HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were grown until confluent and then cell cycle arrested with 24-hour treatment of 2mM thymidine. Migration scratch was then made in the monolayer and capacity of cells to migrate into the free space assessed by light microscopy. Movement of cell migration front was measured as area free of cells and percentage migration calculated and presented graphically. Graphical representation of cell migration is presented as percentage closure of scratch. C. Soft agar colony growth assay Cells were seeded individually in soft agar matrix supplemented with complete growth media and colony formation was assessed every 24 hours over a 6 day period by light microscopy.

Article Snippet: HCT116-GSK3β-KO and paired isogenic control cell line HCT116-GSK3β-WT were created by integrating a ssDNA oligonucleotide containing several stop codons at the GSK3 β locus with TALEN technology (Cellectis).

Techniques: Cell Migration Assay, Migration, Light Microscopy, Growth Assay

A. Map of metabolic pathway perturbations in HCT116-GSK3β-KO cells . All significantly differentially expressed proteins were mapped using the Escher tool for metabolic pathway mapping B. Glycogen metabolism proteins significantly altered in HCT116-GSK3β-KO cells Glycogen metabolism protein sub-network. All red or blue colored proteins were significantly (p<0.05) differentially expressed in wild-type (blue) or knockout cells (red). C. Metabolism phenotype assay of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cell metabolism assessed by Agilent Seahorse XFp cell energy phenotype test, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were used as measures of mitochondrial respiration and glycolysis respectively, under basal and stressed conditions to assess metabolic potential of cells. N=3. D. Growth curve of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cells were seeded at a density of 1 × 10 5 , cell growth was measured using a cell counting chamber every 24 hours, over a period of 9 days. N=3. *= P value <0.05

Journal: bioRxiv

Article Title: Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

doi: 10.1101/2021.01.26.428222

Figure Lengend Snippet: A. Map of metabolic pathway perturbations in HCT116-GSK3β-KO cells . All significantly differentially expressed proteins were mapped using the Escher tool for metabolic pathway mapping B. Glycogen metabolism proteins significantly altered in HCT116-GSK3β-KO cells Glycogen metabolism protein sub-network. All red or blue colored proteins were significantly (p<0.05) differentially expressed in wild-type (blue) or knockout cells (red). C. Metabolism phenotype assay of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cell metabolism assessed by Agilent Seahorse XFp cell energy phenotype test, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were used as measures of mitochondrial respiration and glycolysis respectively, under basal and stressed conditions to assess metabolic potential of cells. N=3. D. Growth curve of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells . Cells were seeded at a density of 1 × 10 5 , cell growth was measured using a cell counting chamber every 24 hours, over a period of 9 days. N=3. *= P value <0.05

Article Snippet: HCT116-GSK3β-KO and paired isogenic control cell line HCT116-GSK3β-WT were created by integrating a ssDNA oligonucleotide containing several stop codons at the GSK3 β locus with TALEN technology (Cellectis).

Techniques: Knock-Out, Cell Counting