Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: The regulation of WDR5 by FBXW7 depends in part on GSK3β. A , FLAG-FBXW7 was overexpressed in HEK293T for 24 h, and all cultures were treated with 5 μM MLN4924 for 5 h prior to harvest. FLAG-FBXW7 was immunoprecipitated, and the indicated sample was dephosphorylated with λ-phosphatase at 30 °C for 3 h. B , GFP-WDR5 was overexpressed in HEK293T for 24 h, and cultures were treated with 5 μM CHIR-99021 or DMSO for 5 h prior to cell harvest, as indicated. GFP-WDR5 was immunoprecipitated using GFP-trap beads. ∗∗ p < 0.01, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , U2OS cells were transfected twice with 30 nM against GL2 or GSK3β and harvested 72 h after the first transfection. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, n = 3. D , GFP-WDR5 and Myc-GSK3β were overexpressed in HEK293T cells for 24 h. GFP-WDR5 was immunoprecipitated using GFP-trap beads. E , the indicated proteins were overexpressed in HEK293T for 24 h. 5 μM CHIR-99021 or 5 μM MLN4924 were added for 9 h and 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. F , FLAG-FBXW7 or FLAG-FBXW7 ARG (R465H, R479Q, and R505C mutations) were coexpressed with Myc-Cullin1 in HEK293T for 48 h and immobilized on α-FLAG M2 beads. In vitro ubiquitination assays of 200 nM GST-WDR5 or GST-c-Myc were performed with 170 nM Uba1, 250 nM of UbcH5b and CDC34, 30 μM ubiquitin, 200 nM His-GSK3β, 5 mM ATP, and immobilized FLAG-FBXW7 constructs at 37 °C for 90 min. DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase-3β; GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Article Snippet: Protein expression was induced by addition of 2 μg/ml doxycycline for at least 72 h. GSK3β activity was blocked with 5 μM CHIR-99021 (MedChemExpress; catalog no.: HY-10182) and cullin-RING E3 ubiquitin ligases with 5 μM MLN4924 (Cell Signaling Technology; catalog no.: 85923) for 5 h prior to cell harvest.

Techniques: Immunoprecipitation, Transfection, In Vitro, Ubiquitin Proteomics, Construct

Journal: Translational Oncology

Article Title: Cinobufacini enhances the therapeutic response of 5-Fluorouracil against gastric cancer by targeting cancer stem cells via AKT/GSK-3β/β-catenin signaling axis

doi: 10.1016/j.tranon.2024.102054

Figure Lengend Snippet: Cinobufacini suppresses β-catenin activity by modulating AKT/GSK-3β signaling in GC cells. (A) Protein level of AKT, p-AKT,GSK-3β, p-GSK-3β, total β-catenin and non-phosphorylated (Ser33/37/Thr41) β-Catenin (Active) was detected by western blotting in MKN-45 and SGC-7901 cells after Cinobufacin treatment. α-TUBLIN protein serves as the internal parameter. The gray value is quantified using ImageJ software and analyzed statistically. ( n = 3, mean ± SD. * p < 0.05 and ** p < 0.01 compared with the control group.). (B) The luciferase reporter assay was employed to evaluate the activity of the TCF/LEF-controlled luciferase reporter in MKN-45 cells treated with or without Cinobufacini and/or CHIR99021.(C) The relative mRNA expression levels of CCND1 and MYC were detected by qRT–PCR in MKN-45 cells with or without the Cinobufacini or/and CHIR99021 treatment. ( n = 3, mean ± SD. * p < 0.05 and ** p < 0.01 compared with the control or DMSO). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cinobufacini injection was acquired from Anhui Huarun Jinchan Pharmaceutical Co. Ltd. 5-FU (HY-90006) and GSK-3β inhibitors CHIR-99021 (HY-10182B) was purchased from MedChemExpress Company.

Techniques: Activity Assay, Western Blot, Software, Control, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: Cinobufacini enhances the therapeutic response of 5-Fluorouracil against gastric cancer by targeting cancer stem cells via AKT/GSK-3β/β-catenin signaling axis

doi: 10.1016/j.tranon.2024.102054

Figure Lengend Snippet: Cinobufacin suppresses the maintenance of GCSCs and inhibits the progression of GC in transplanted tumors by modulating β-Catenin signaling (A-B) The immunohistochemistry staining of the total β-Catenin and active β-Catenin protein accumulated in tumor tissues from MGC—CSC xenografts (Scale bar = 20 μm). (B) To ensure accuracy and reliability, statistical analysis was performed to quantify the protein levels of total β-Catenin and active β-Catenin in (A). ( n = 3, mean ± SD. * p < 0.05 and ** p < 0.01 compared with the control or 5-FU group. (C) The immunofluorescence staining was conducted to mark the p-GSK-3β (red), total β-catenin (green), CD44 (purple) and nucleus (DAPI, blue) in tumor tissue (Scale bar = 20 μm). (D) The relative mRNA expression levels of Wnt/β-Catenin targets ( CCND1 and MYC ), core stemness factors ( NANOG, SOX2 , and OCT4 ) and CSCs markers ( LGR5 and CD44 ) in tumor tissue were detected by qRT–PCR ( n = 6, mean ± SD. * p < 0.05 and ** p < 0.01 compared with the control or 5-FU group.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cinobufacini injection was acquired from Anhui Huarun Jinchan Pharmaceutical Co. Ltd. 5-FU (HY-90006) and GSK-3β inhibitors CHIR-99021 (HY-10182B) was purchased from MedChemExpress Company.

Techniques: Immunohistochemistry, Staining, Control, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Nature

Article Title: Phenotypic landscape of intestinal organoid regeneration

doi: 10.1038/s41586-020-2776-9

Figure Lengend Snippet: a, An example image (MIP of a confocal z -stack) of an organoid, with annotated regions along the major axis, and showing antibody staining for lysozyme (Lyz) and aldolase B (Aldob). Lysozyme-positive Paneth cells are indicated by white arrowheads. Scale bar, 50 μm. b , Left, an example image of a stitched well-level overview. Scale bar, 100 μm. Middle, enlarged regions from the left panel. Scale bar, 100 μm. Right, segmentation masks, with object unique identifier numbers overlaid. a, b , Representative images of the DMSO control condition, repeated in n = 471 independent replicates with similar results. c, Top, reproducibility of detected organoid counts in replicas of the library plates. Heat map of Pearson’s correlation of organoid counts in screen replicates, with plate replicates grouped by parent library plates as indicated; n = 384 wells per plate. Bottom, heat map of standard deviation of organoid size ( z -scored organoid area) for respective well positions. Passive and active controls are in wells outlined by the white box. n = 18 independent plates. d , Left, covariance matrix of the 34 extracted features, clustered by covariance (c-means clustering, ten clusters). Ch, channel; Stdlntensity, standard deviation of intensity. Right, heat map showing principal component loading of the extracted features; bar chart above, eigenvalues of first ten principal components. e, Top, changes in the accuracy of the naive Bayes predictor throughout the backwards-feature- elimination test. Bottom, final set of nine features (‘Algorithm-selected’) and list of features used as input for clustering with PhenoGraph. f , Distribution of mean values per well of the indicated features in active and passive control conditions. CHIR is the GSK3B inhibitor CHIR99021; DAPT is a γ-secretase inhibitor. Centre line, median; boxes, quartile range; individual data points overlaid. Two-sided t-test, with P-values shown in the plot. MeanInt, mean intensity. n = 471, 100 and 101 independent samples for the DMSO, CHIR99021 and DAPT conditions respectively. g , Z-scored organoid features used for clustering, shown for individual well positions of in-plate replicates of active and passive control conditions. n = 6, 6 and 20 wells per plate for DAPT, CHIR and DMSO respectively; shown are mean values over n = 18 assay plates. D, DAPI; Eccentr., eccentricity, Integr. Int., integrated intensity; Mass disp., mass displacement for the indicated immunofluorescence channels; Std Int., standard deviation of intensity. h , Mean z -scored values for the indicated features per well from the entire dataset, sorted in ascending order. Every data point is an average value per well, with conditions colour-coded as in the key. n = 5,250 independent samples measured in the screen. i , tSNE maps colour coded for z -scored values of indicated features (left to right: eccentricity, organoid area, mean intensity of aldolase B and mean intensity of lysozyme antibody staining) and PhenoGraph classification. Every data point represents an individual organoid, with sampling stratified by PhenoGraph assignment. The sample size is 5,000 individual organoids.

Article Snippet: From day 0 to day 3, WENRC medium (ENR medium supplemented with 20% Wnt3a-conditioned medium (Wnt3a-CM), 10 μM ROCK inhibitor Y-27632 (Stem Cell Technologies) and 3 μM GSK-3β inhibitor CHIR99021 (Stem Cell Technologies, catalogue number 72054)) was used for organoid culture.

Techniques: Staining, Control, Standard Deviation, Immunofluorescence, Sampling

Journal: Nature

Article Title: Phenotypic landscape of intestinal organoid regeneration

doi: 10.1038/s41586-020-2776-9

Figure Lengend Snippet: a, Top, phenotypic clusters and phenotypic groups (see ) identified using the multivariate feature array, and heat map of mean z-scored values per class for the indicated feature array. Aldob, aldolase B; Ar, area; D, DAPI; Ecc, eccentricity; II, integrated intensity; Lyz, lysozyme; MD, mass displacement; MI, mean intensity; SI, standard deviation of intensity; Sh, shape. n = 40,624, 34,265, 20,134, 31,687, 2,549, 702, 23,643, 48,992, 30,791, 21,342, 23,610, 32,773, 19,172, 41,931 and 29,939 organoids for classes 1-15 respectively. Bottom, phenotypic class assignment for organoids in control conditions; outer circle colour, phenotypic group; inner circle colour, assigned phenotypic cluster; n = 7367, 7982 and 33395 individual organoids for DAPT, CHIR99021 (CHIR) and DMSO conditions respectively. b , Potential loss of phenotypic resolution owing to phenotype grouping. Three phenotypic clusters are assigned to the ‘regenerative’ phenotype, but the exact 15-element phenotypic signature differs depending on whether PORCN or GSK3β is inhibited. Mean of n = 100, 4, 2 and 2 independent replicates for CHIR99021, Cpd2170, Cpd420 and Cpd1772 respectively. c, Top, relative z-score transformed abundances of 15 phenotypic clusters, shown as means ± s.d. Bottom, heat maps showing individual and mean phenotypic signatures for 50 replicates of the indicated control conditions. n = 50 sampled independent replicates for all 3 conditions. d , Left, randomization trial for reshuffling of phenotypic class labels in the entire dataset. Right, scatter plots and histograms showing the distribution of replica correlation and maximum class variance in permuted and non-permuted data for the selected and filtered conditions. Red dashed lines show the original selection stringency. e , Distribution of calculated P-values in the indicated groups of conditions. Top, scatter plot and kernel density estimation; bottom, box plots. Boxes, quartile range; whiskers, value interval with excluded outliers; solid lines, median values. n = 2,242 unique conditions corresponding to 5,204 individual wells. n = 279, 15 and 1,948 conditions for ‘hit’, ‘hit (one replica)’ and ‘not selected’ groups respectively. f , Top left, mean z -scored values per identified phenotypic class for the indicated feature array. Bottom left, heat map representation of the phenotypic signature for the indicated compounds, grouped by the most abundant phenotype in the original screen. Top right, tSNE map colour coded for the highest enriched phenotypic clusters. Every data point represents a compound. tSNEs were calculated from phenotypic signatures. Bottom, calculated reproducibility values per compound and numbers of replicates used for analysis of the indicated compounds. The replica count is the number of non-sparse (n > 10 organoids) independent wells recorded per condition. Assay performed in n = 8 independent replica from 2 independent organoid cultures. g , Flowchart depicting filtering of the screened conditions. h , tSNE maps colour coded for the highest enriched phenotypic clusters as indicated; every data point represents either a compound condition (top; n = 301 compound conditions) or a unique target gene-modality pair (bottom; n = 207 target genes); tSNEs calculated from 15-dimensional phenotypic signatures.

Article Snippet: From day 0 to day 3, WENRC medium (ENR medium supplemented with 20% Wnt3a-conditioned medium (Wnt3a-CM), 10 μM ROCK inhibitor Y-27632 (Stem Cell Technologies) and 3 μM GSK-3β inhibitor CHIR99021 (Stem Cell Technologies, catalogue number 72054)) was used for organoid culture.

Techniques: Standard Deviation, Control, Transformation Assay, Selection

Journal: Cancers

Article Title: Differential Transcriptomic Profiles Following Stimulation with Lipopolysaccharide in Intestinal Organoids from Dogs with Inflammatory Bowel Disease and Intestinal Mast Cell Tumor

doi: 10.3390/cancers14143525

Figure Lengend Snippet: The composition of complete chelating solution (CCS) and organoid culture media.

Article Snippet: * Aminopyrimidine CHIR99021 (GSK3β inhibitor) , 2.5 μM , Reprocell.

Techniques: Concentration Assay, Recombinant