gsk-3β inhibitor chir99021  (Tocris)

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: A Immunoblot showing HuR levels in HuR RIP performed from K562 cells. B Graph summarising the fold enrichment ACTB mRNA (known HuR binding target) obtained from RT-qPCR analysis of IgG or HuR RIP analysis from K562 cells ( n = 3) C Representative immunoblot showing β-catenin level obtained from β-catenin RIPs performed from K562 cells (±CHIR99021), ID = immunodepleted lysate. D Agilent 2100 Bioanalyzer gel showing RNA isolated from IgG or β-catenin RIP performed from K562 cells (±CHIR99021).

Article Snippet: The myeloid cell lines K562, HL60, HEL, U937, PLB-985, NOMO1, OCI-AML3, EOLI, ML-1, THP-1, KU812 (The European Collection of Authenticated Cell Cultures) and OCI-AML2, MV4-11, KG-1, KG1a SET2, NB4 and MONOMAC6 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were confirmed mycoplasma-free and authenticated via short-tandem repeat (STR) analysis prior to general culture as previously [ ]. β-Catenin was stabilised using the GSK-3β inhibitor CHIR99021 (Merck-Millipore) as previously described [ ], whilst transcription was inhibited via 2 μg/ml Actinomycin D (Merck-Millipore) at the stated timepoints.

Techniques: Western Blot, Binding Assay, Quantitative RT-PCR, Isolation

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: Volcano plots showing the fold change in mRNA abundance detected within β-catenin RIP performed from DMSO (basal Wnt signalling) versus CHIR99021 (activated Wnt signalling) treated A K562 or B HEL cells ( n = 3). Red dots represent enriched ( P adj < 0.05) mRNAs whilst blue dots highlight Wnt signalling mRNAs and black dots highlight metabolic mRNAs. C Venn diagram illustrating the unique and shared mRNAs partners identified from β-catenin RIP performed from CHIR99021 versus DMSO treated K562 or HEL cells. D Gene ontology (GO) analysis using the human Molecular Signatures Database (Elsevier Pathway Collection) via Enrichr for pathways represented amongst the most significantly and commonly enriched mRNAs ( P adj < 0.05), obtained in β-catenin RIP from K562 and HEL cells ±CHIR99021 with adjusted −Log 10 P values annotated. Summary graphs showing the fold enrichment of selected Wnt signalling mRNAs isolated from IgG or β-catenin RIP-RT-qPCR performed in E K562 and F HEL cells. Data represents mean ± 1 s.d ( n = 3). Statistical analysis is denoted by * p < 0.05 and ** p < 0.01 as deduced from a one-sample t-test.

Article Snippet: The myeloid cell lines K562, HL60, HEL, U937, PLB-985, NOMO1, OCI-AML3, EOLI, ML-1, THP-1, KU812 (The European Collection of Authenticated Cell Cultures) and OCI-AML2, MV4-11, KG-1, KG1a SET2, NB4 and MONOMAC6 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were confirmed mycoplasma-free and authenticated via short-tandem repeat (STR) analysis prior to general culture as previously [ ]. β-Catenin was stabilised using the GSK-3β inhibitor CHIR99021 (Merck-Millipore) as previously described [ ], whilst transcription was inhibited via 2 μg/ml Actinomycin D (Merck-Millipore) at the stated timepoints.

Techniques: Isolation, Quantitative RT-PCR

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: A Scatter plots showing MSI2 detection in β-catenin interactomes performed in cytosolic fractions of K562 and HEL cells. Vertical dashed red line indicates the threshold for 2-fold change in protein binding at log 2 (=1) relative to IgG Co-IP. The horizontal red line represents threshold for p < 0.05 on log 10 scale (=1.3). Highlighted red dots indicate enriched interactions ( p < 0.05), green labels highlight position of MSI2, and blue labels highlight position of β-catenin bait. Representative immunoblots showing the level of β-catenin protein present in MSI2 Co-IPs derived from B K562 - RNase A, C K562 + 20 µg/mL RNaseA, D HEL - RNase A and E HEL + 20 µg/mL RNaseA whole cell lysates, ±5µM CHIR99021 overnight. ID immunodepleted lysate.

Article Snippet: The myeloid cell lines K562, HL60, HEL, U937, PLB-985, NOMO1, OCI-AML3, EOLI, ML-1, THP-1, KU812 (The European Collection of Authenticated Cell Cultures) and OCI-AML2, MV4-11, KG-1, KG1a SET2, NB4 and MONOMAC6 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were confirmed mycoplasma-free and authenticated via short-tandem repeat (STR) analysis prior to general culture as previously [ ]. β-Catenin was stabilised using the GSK-3β inhibitor CHIR99021 (Merck-Millipore) as previously described [ ], whilst transcription was inhibited via 2 μg/ml Actinomycin D (Merck-Millipore) at the stated timepoints.

Techniques: Protein Binding, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: A Immunoblots showing MSI2, β-catenin and LEF1 level in K562 and KU812 cells harbouring MSI2 shRNA or non-targeting shRNA controls. GAPDH indicates protein loading. B Representative flow cytometric histograms showing intensity of the TCF-dependent expression of Venus Yellow Fluorescent Protein (YFP) from the β -catenin a ctivated r eporter (BAR) reporter, or negative control ‘ f ound u nresponsive’ BAR (fuBAR; containing mutated promoter binding sites) in K562 and KU812 cells ± MSI2 shRNA following treatment with 5 μM CHIR99021 overnight. The fuBAR (dashed), non-targeting control shRNA (grey filled), and two MSI2 shRNAs (blue or red) histograms are shown. Summary graphs showing the median fluorescence intensity (MFI) generated from the BAR/fuBAR in C K562 and D KU812 cells ±MSI2 shRNA with ±5 μM CHIR99021. E Immunoblots showing total β-catenin, LEF-1, and MSI2 subcellular localisation in K562 and KU812 cells lentivirally transduced with two different MSI2 shRNAs ±5 μM CHIR99021. Lamin A/C and α-tubulin indicate the purity/loading of the nuclear (N) and cytosol (C) fractions respectively. Densitometric quantitation of LEF-1 (relative to nuclear Lamin A/C, AU = arbitrary units) present in nuclear fractions of F K562 and G KU812 cells ± MSI2 shRNA with ±5 μM CHIR99021. Summary graph showing the fold change in LEF1 and TCF7L2 mRNA expression as assessed by RT-qPCR in ( H ) K562 and ( I ) KU812 cells expressing MSI2 shRNA relative to non-targeting control shRNA (represented by dotted line at y = 1). Fold change is relative to matched respective controls (black dashed line) and overall expression was normalised to the housekeeping gene β-actin ( ACTB ). Data represent mean ± 1 s.d ( n = 3). Statistical analysis is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as deduced from a student’s t test or one-sample t-test for RT-qPCR data.

Article Snippet: The myeloid cell lines K562, HL60, HEL, U937, PLB-985, NOMO1, OCI-AML3, EOLI, ML-1, THP-1, KU812 (The European Collection of Authenticated Cell Cultures) and OCI-AML2, MV4-11, KG-1, KG1a SET2, NB4 and MONOMAC6 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were confirmed mycoplasma-free and authenticated via short-tandem repeat (STR) analysis prior to general culture as previously [ ]. β-Catenin was stabilised using the GSK-3β inhibitor CHIR99021 (Merck-Millipore) as previously described [ ], whilst transcription was inhibited via 2 μg/ml Actinomycin D (Merck-Millipore) at the stated timepoints.

Techniques: Western Blot, shRNA, Expressing, Negative Control, Binding Assay, Control, Fluorescence, Generated, Transduction, Quantitation Assay, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocol to engineer apical-out airway organoids using suspension culture of human airway basal stem cell aggregates

doi: 10.1016/j.xpro.2023.102154

Figure Lengend Snippet:

Article Snippet: CHIR99021 (GSK-3β inhibitor), 10 mM , Reprocell , Cat# 04000402.

Techniques: Recombinant, Cell Culture, Saline, Sterility, Pore Size, Transferring, Microscopy, Blocking Assay

Journal: STAR Protocols

Article Title: Protocol to engineer apical-out airway organoids using suspension culture of human airway basal stem cell aggregates

doi: 10.1016/j.xpro.2023.102154

Figure Lengend Snippet: Complete Airway Basal Stem Cell Expansion Medium

Article Snippet: CHIR99021 (GSK-3β inhibitor), 10 mM , Reprocell , Cat# 04000402.

Techniques: Concentration Assay