β-catenin-responsive reporter gene (topflash-luc)  (Promega)

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: p38α is a molecular partner of the cytoplasmic β-catenin destruction complex. Co-immunoprecipitation assays showing that endogenous p38α is a molecular partner of APC, Axin1, β-catenin, and GSK3β in normal colon tissue from C57BL/6 mice and HCT-116 CRC cells. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Immunoprecipitation

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Effect of p38α inhibition in a CRC mouse model. A Mice treatment scheme. APC Min/+ mice were administered with AOM (14 mg/kg body weight) once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190 (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed. B Graph showing the reduction in the number of colon tumors in animals treated with SB202190. Tumors were stained with methylene blue, counted, and measured. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. DMSO. C Immunohistochemical analysis of p38α and β-catenin cytoplasmic and nuclear staining in C57BL/6 mice and AOM-treated APC Min/+ mice injected with the p38α inhibitor SB202190 or DMSO. Original magnification: 100 × and 200 × . p38αi = p38α inhibitor. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Inhibition, Staining, Immunohistochemistry, Injection

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Uncoupling p38α cytoplasmic and nuclear functions in the Wnt pathway. A , H Immunoblotting analysis of p38α and β-catenin cellular localization in HT-29 ( A ) and HCT-116 ( H ) CRC cells under serum starvation (24 h) and upon activation of the Wnt pathway mediated by serum supplementation ( A , H ) or addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h ( A ). Subsequently, cells were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h ( A , H ). B – F Densitometric analysis of the indicated protein levels against the loading control in the different culture conditions used in this study. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. no serum ( B , C ) or vs. no ralimetinib ( D – F ). G RTqPCR analysis of β-catenin target gene expression in HT-29 cells treated as in A . Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. no serum; # P < 0.05 vs. no ralimetinib. Lamin B1: nuclear loading control; PDI: cytoplasmic loading control. N = Nucleus, C = Cytoplasm. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Western Blot, Activation Assay, Expressing

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Immunofluorescence analysis of p38α and β-catenin cellular localization. Immunofluorescence analysis of p38α and β-catenin cellular localization in HT-29 CRC cells under serum starvation (24 h) and upon activation of the Wnt pathway by serum supplementation or addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Immunofluorescence, Activation Assay

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: p38α modulate β-catenin target gene expression. A Chromatin immunoprecipitation assays of Wnt target genes in HT-29 cells under serum starvation (24 h) and upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. Quantification was done using the % input method. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. untreated cells, and # P < 0.05 vs. no ralimetinib. B RTqPCR analysis of Wnt target genes in HT-29 cells upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h after p38α genetic ablation for 24 h or as a pre-treatment before p38α inhibition with ralimetinib (10 μM) for 24 h. Data are presented as mRNA fold change vs. control. The dotted line corresponds to the expression levels detected in control conditions (siRNA CTRL/DMSO). Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. siRNA CTRL/DMSO. C TOPFlash/FOPFlash assay for Wnt transcriptional activity. HT-29 cells were first transfected to overexpress p38α and β-catenin; after 24 h, cells were transfected with TOP/FOP plasmids, serum-starved for 24 h and then stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with ralimetinib (10 μM) for 24 h. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. empty vector, # P < 0.05 vs. DMSO, ▲ P < 0.05 vs. no serum. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Expressing, Chromatin Immunoprecipitation, Activation Assay, Inhibition, Activity Assay, Transfection, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Characterization of p38α kinase activity on β-catenin. A In vitro kinase assay showing β-catenin phosphorylation by p38α in the absence or presence of ralimetinib at the indicated concentrations. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. active p38α; Δ P < 0.05 vs. active p38α + β-catenin. B MS/MS spectrum of the double-charged precursor ion of peptide A96AMFPETLDEGMQIPS111T112QFDAAHPTNVQR124. C In vitro kinase assay showing phosphorylation of β-catenin-WT, β-catenin-S111A, and β-catenin-T112A by p38α. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Activity Assay, In Vitro, Kinase Assay, Tandem Mass Spectroscopy

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: The identified β-catenin residues targeted for phosphorylation by p38α are crucial for β-catenin transcriptional activity. A Co-immunoprecipitation of p38α with FLAG-β-catenin-WT or FLAG-β-catenin-S111A or FLAG-β-catenin-T112A in HCT-116 cells. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. B Chromatin immunoprecipitation assay in HCT-116 cells. Cells overexpressing FLAG-β-catenin-WT or FLAG-β-catenin-S111A or FLAG-β-catenin-T112A were treated or not with ralimetinib (10 μM) for 24 h. Chromatin was pulled down with anti-FLAG antibodies. Anti-IgGs were used as negative controls. *P < 0.05 vs. FLAG-β-catenin-WT; # P < 0.05 vs. DMSO. C Quantification results of the ddPCR assay (copies/µL) of β-catenin and c-Myc mRNA expression, as processed by QuantaSoft. HCT-116 CRC cells silenced by genetic ablation for endogenous β-catenin and exogenously expressing β-catenin-WT, β-catenin-S111A, β-catenin-T112A were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. The error bars represent the maximum and minimum Poisson distribution for the 95% confidence interval generated by QuantaSoft. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Activity Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Expressing, Generated

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Pharmacological targeting of p38α inhibits β-catenin transcriptional activity in patient-derived CRC-SCs and tumor intestinal organoids. A Schematic representation of the experimental procedure used for generating patient-derived CRC-SCs and organoids (created with BioRender.com). B Quantification results of the digital droplet PCR (ddPCR) assay (copies/µL) of c-Myc mRNA expression, as processed by QuantaSoft. Patient-derived CRC-SC tumorspheres (left panel) and patient-derived CRC organoids (right panel) were treated with the GSK3β inhibitor TWS-119 (10 μM) for 4 h and subsequently treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. The error bars represent the maximum and minimum Poisson distribution for the 95% confidence interval generated by QuantaSoft. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Activity Assay, Derivative Assay, Expressing, Generated

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: Schematic representation of p38α and β-catenin localization and activity under the different experimental conditions used in this study. GSK3βi GSK3β inhibitor, p38αi p38α inhibitor

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Activity Assay