Journal: National Science Review

Article Title: Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos

doi: 10.1093/nsr/nwad173

Figure Lengend Snippet: Wnt inhibition greatly improves the development of SCNT embryos. (a) Comparison of Wnt-related genes in differential gene expression profiles of the ICM of IVF and SCNT blastocysts. The genes marked are Wnt antagonists or activators. (b) TCF/Lef: H2B-GFP IVF (left) and SCNT (right) embryos at late E4.5 stained for GFP, Oct4, Gata6 and DAPI. Scale bar, 20 μm. (c) Percentage of GFP-positive cells in the EPI of IVF and SCNT embryos. (d) Percentage of rosette-like SCNT embryos generated with no treatment, Wnt activator CHIR treatment and Wnt inhibitor IWP2 treatment. (e) Comparison of pluripotent marker gene expression among the IVF-, SCNT- and Wnti-treated SCNT embryos. (f) Absolute value comparison of the relative expression of markers in (d) to late-E4.5 IVF EPI between groups of IVF-, SCNT- and Wnti-SCNT embryos. P -values were determined using the Wilcoxon signed-rank test. (g) GSEA of specific genes expressed in naïve and primed ESCs between the late-E4.5 EPI of untreated and Wnti-SCNT embryos. NES, normalized enrichment score. (h) PCA comparison of gene expression profiles among the late-E4.5 EPI of IVF-, SCNT- and Wnti-SCNT embryos, naïve ESCs, primed ESCs and RSCs. Published transcriptome data of stem cell lines were obtained from GSE145727. (i) Hierarchical clustering analysis of the late-E4.5 EPI of IVF, untreated and Wnti-SCNT embryos, naïve ESCs, primed ESCs and RSCs. (j) PCA plot of E3.5 ICM, E5.5 EPI, E6.5 EPI, late-E4.5 EPI of IVF, SCNT and Wnti-SCNT embryos based on the H3K27me3 levels. (k) Heat map showing the H3K27me3 levels for the naïve and primed markers between late-E4.5 EPI of IVF, SCNT and Wnti-SCNT embryos. For (c) and (d), P -values were determined using the unpaired two-tailed t -test; error bars and means ± SD are shown for n ≥ 3 experiments.

Article Snippet: Small-molecule inhibitors IWP2 (Selleck, 686770-61-6), IWR1-endo (Selleck, 1127442-82-3) and Wnt activator CHIR99021 (Sigma, SML1046) were applied to inhibit or activate the canonical Wnt/β-catenin signaling pathway.

Techniques: Inhibition, Comparison, Gene Expression, Staining, Generated, Marker, Expressing, Two Tailed Test

Journal: Cell death discovery

Article Title: Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells.

doi: 10.1038/s41420-023-01616-6

Figure Lengend Snippet: Fig. 2 Transcriptome analysis of differentiated hiPSCs (IMR90) toward CMs. The hiPSCs were cultured as a monolayer on matrigel-coated plates for 2 days under pluripotent conditions and on day 0 exposed to GSK3 inhibitor, CHIR (10 µM) for 24 h. After 48 h exposed to Wnt inhibitor, IWP2 (5 µM). Spontaneously beating cardiac clusters were observed from day 9 onwards. Simultaneously, cells were exposed to test substances for a single exposure of 24 h (day1). The cells were harvested for gene array analysis on day1, day4 and day14 (Fig. 1A). Medium changes were done as indicated every alternate date. A Representative phase-contrast images of control and ISO treated hiPSC at day1-, 4 -and 14day. Scale bar, 100 µm. B PCA blot of 54,675 probe sets for three timepoints during the differentiation. C PCA blot of the 500 SPS with the highest variance across the mean of the condition‐wise samples. The respective day is indicated by the shape and the respective measured compound is indicated by the color of the dot, as labels are shown next to the plots. The distribution of the data points on the x-axis is given by the PC 1 and on the y-axis by PC2. The percentages in parentheses denote the proportion of explained variance for the respective PC.

Article Snippet: The medium was then changed to basal RPMI/B-27-ins medium and cells were kept for further 24 h. At day 2, RPMI/B-27-ins mediumwith small molecule WNT inhibitor IWP2 (Tocris, United Kingdom) 5 μM was added and cells were kept for 48 h (day 2 to day4).

Techniques: Cell Culture, Control

Journal: mSphere

Article Title: Wnt5A Signaling Regulates Gut Bacterial Survival and T Cell Homeostasis

doi: 10.1128/msphere.00507-22

Figure Lengend Snippet: Wnt5A signaling influences commensal bacterial survival in the Peyer’s patches. (A and B) Wnt5A expression in total cells of Peyer’s patches of BALB/c mice by immunoblot (A) and ELISA of cell supernatant (B) ( n = 2 to 3). (C) Intracellular bacteria in the adherent cells of Peyer’s patches as observed by fluorescence microscopy after PI staining. (D) Estimation of Enterococcus spp. and Lactobacillus spp. in Peyer’s patches by qPCR ( n = 4 mice); NTC indicates no-template control, and “culture” ( E. faecalis or L. rhamnosus ) is used as the positive control. (E and F) Application of Dsh and Wnt5A production inhibitor IWP2 for 6 h led to diminution of countable bacterial CFU from total cells (E) and adherent cells (F) of Peyer’s patches ( n = 3 to 4). (G) Bacteria (PI, red), denoted by arrowhead, associated with CD11b+ (green) phagocytes of Peyer’s patches as observed by confocal microscopy. (H) IWP2 blocked survival of E. faecalis and L. rhamnosus in adherent cells of Peyer’s patches as observed from CFU 3 h ( T 3) postinternalization ( T 0) ( n = 4). (I) Administration of Arp 2/3 complex inhibitor I (CK666) and Arp 2/3 complex inhibitor II (CK869) for 6 h to cells of Peyer’s patches led to a decrease in endogenous CFU ( n = 3). Data are represented as the mean ± SEM, and a P value of ≤0.05 was considered statistically significant. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.

Article Snippet: IWP2 , Santa Cruz Biotechnology, USA , SC-252928.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Bacteria, Fluorescence, Microscopy, Staining, Control, Positive Control, Confocal Microscopy

Journal: mSphere

Article Title: Wnt5A Signaling Regulates Gut Bacterial Survival and T Cell Homeostasis

doi: 10.1128/msphere.00507-22

Figure Lengend Snippet: Adaptation of gut commensal bacterial colonization to changes in Wnt5A is linked with altered T cell activation profiles. (A) Colocalization of CD4 + T cells (green) and CD11c+ phagocytes (red) in Peyer’s patch tissue visualized by confocal microscopy. The inset shows colocalization (yellow) of both cell types ( n = 4 mice). (B) FACS representing the difference between the IL17a and FoxP3 expressing CD4 + T cell populations in Peyer’s patches of wild-type and heterozygous mice. (C) Plot showing a higher IL17a/FoxP3 ratio, as well as a higher number of IL17a+FoxP3+ CD4 T cells in Peyer’s patches of heterozygous mice lacking a functional copy of the Wnt5A gene as opposed to the wild type ( n = 6 mice). (D) FACS representing the difference in IL17a+- and FoxP3+-expressing CD4 T cell populations in total cells of Peyer’s patches where Wnt5A signaling was downregulated by IWP2 treatment for 6 h. (E) Plot showing that IWP2 treatment increased the IL17a/FoxP3 ratio and number of IL17a+FoxP3+ CD4 T cells in the total cell population of Peyer’s patches ( n = 5). (F and G) Higher MHC II surface expression in both CD11b+ (F) and CD11c+ (G) phagocytes of Peyer’s patches upon inhibition of Wnt5A signaling by IWP2 ( n = 5). Marker M1 indicates the percentage of CD11b+ or CD11c+ cells that were positive for MHC II expression. The markers, quadrants, and gates used for identification were based on the unstained population. Data are represented as the mean ± SEM. A P value of ≤0.05 was considered statistically significant. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.

Article Snippet: IWP2 , Santa Cruz Biotechnology, USA , SC-252928.

Techniques: Activation Assay, Confocal Microscopy, Expressing, Functional Assay, Inhibition, Marker

Journal: mSphere

Article Title: Wnt5A Signaling Regulates Gut Bacterial Survival and T Cell Homeostasis

doi: 10.1128/msphere.00507-22

Figure Lengend Snippet: List of reagents used in the study ^a

Article Snippet: IWP2 , Santa Cruz Biotechnology, USA , SC-252928.

Techniques: cDNA Synthesis, Membrane, Control, Staining, Cell Isolation