Journal: Frontiers in Immunology

Article Title: Meta-analysis of multi-center transcriptomic profiles and machine learning reveal phospholipase Cβ4 as a Wnt/Ca² + signaling mediator in glioblastoma immunotherapy

doi: 10.3389/fimmu.2025.1610683

Figure Lengend Snippet: In vitro experiments showing that PLCB4 in the ProImmuML signature may be a downstream regulator of the Wnt pathway and inhibit GBM proliferation. (A) Metascape analysis of the ProImmuML signature. (B) qPCR analysis was performed to analyze the effects of three different Wnt pathway agonists/inhibitors on the expression of APCDD1 (left panel), RAC2 (middle panel), and PLCB4 (right panel). SKL: SKL-2001, Wnt/β-catenin signaling pathway agonist; MSAB: MSAB, Wnt/β-catenin signaling pathway inhibitor; 2-APB: 2-APB, Wnt/Ca 2+ signaling pathway inhibitor; LON: Lonomycin, Wnt/Ca 2+ signaling pathway agonist; BLE: Blebbistatin, Planar cell polarity pathway inhibitor; WNT: Wnt5a, Planar cell polarity pathway agonist. (C) Representative IHC staining images of PLCB4 from four glioma patients with higher expression of PLCB4 (left panel, n = 2) and lower expression of PLCB4 (right panel, n = 2). (D) The intersection of genes from two parallel comparisons identified 235 upregulated genes (left panel) and 65 downregulated genes (right panel). (E) KEGG enrichment analysis of the upregulated genes. (F) GSEA revealing proliferation-related pathways were significantly inhibited after overexpressing PLCB4. (G) EdU assay showing PLCB4 inhibiting the proliferation of glioma cells in U87. Significant difference, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Lonomycin (LON; TargetMol, China) and 2-APB (TargetMol, China) served as the activator and inhibitor of the Wnt/Ca2 + pathway, while Wnt5a (TargetMol, China) and blebbistatin (TargetMol, China) were used to modulate the Wnt/PCP pathway.

Techniques: In Vitro, Expressing, Immunohistochemistry, EdU Assay

Journal: BMC cancer

Article Title: ATBF1 is a potential diagnostic marker of histological grade and functions via WNT5A in breast cancer.

doi: 10.1186/s12885-022-10380-2

Figure Lengend Snippet: Fig. 8 WTN5A was a crucial downstream gene of ATBF1. A Venn diagram of genes involved in the top 3 enriched GO (cell communication, extracellular matrix and system development) and the top 1 enriched KEGG (pathways in cancer). Five common genes (WNT5A, WNT6, WNT11, WNT5B and LAMA1) were detected. B The five common genes were listed by the fold change. The color represents the P value. C The expression of WNT5A in MCF7 cells transfected with siControl or siATBF1 was examined by real-time qPCR and western blot. Down-regulation of WNT5A was observed after ATBF1 silencing. Triplicate experiments were performed and P-value between two groups was calculated by student’s t test. D The mRNA expression of WNT5A in paired breast cancer cases, as determined by real-time qPCR. E The protein levels of WNT5A in paired breast cancer cases, quantified by WI index. Paired t test was used to calculate P-value in Fig. 8D&E. F Representative IHC images of WNT5A in paired breast cancer cases. G The correlation between the expression of WNT5A and ATBF1 at protein level, quantified by WI index. H Cell proliferation assay. MCF7 cells were transfected with siControl or siATBF at the concentration of 150 nM. Twenty four hours later, the cells were treated with 200ng/mL recombinant human WNT5A to rescue the down-regulation of WNT5A induced by ATBF1 knockdown. The cell number was determined as OD450 after 24 h of WNT5A treatment with a cell counting kit. One-way ANOVA with Bonferroni was used to determine the statistical differences among the three groups

Article Snippet: The reagents were purchased from their respective vendors: expose mouse- and rabbit-specific HRP/ DAB detection IHC kit (Cat. ab236466, Abcam, Shanghai, China); RNA-Solv Reagent (Cat. R6830, Omega, Guangzhou, China); TRIzolTM (Cat. 15,596,026, Invitrogen, Shanghai, China); BCA Protein Assay Kit (Cat. CW0014S, CWBIO, Jiangsu, China); secondary antibodies (BOSTER, Wuhan, China); Lipofectamine RNAiMAX reagent (Cat. 13,778,100, Invitrogen); recombinant human WNT5A (Cat. CSB-EP026138HU1, CUSABIO, Wuhan, China); cell counting kit (APE×Bio, Shanghai, China); WNT5A primary antibody (Cat. A19133, Abclonal, Wuhan, China); and β-actin primary antibody (Cat. AC026, Abclonal).

Techniques: Expressing, Transfection, Western Blot, Proliferation Assay, Concentration Assay, Recombinant, Knockdown, Cell Counting

Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA-seq) analysis of the dynamically cultured EO and HEO complexes. a. UMAP visualization of 12 samples across four culture conditions: HEOe (HEO + estrogen), HEOp (HEO + estrogen + progesterone + cAMP), EOe (EO + estrogen), EOp (EO + estrogen + progesterone + cAMP). Endometrial epithelial cell clusters were annotated as main Epi (mEpi), ribosome-related Epi (rEpi), proliferative Epi (pEpi), and ciliated Epi (cEpi). Endometrial stromal cell clusters were annotated as main Str (mStr), ribosome-related Str (rStr), proliferative Str (pStr), and a second proliferative Str cluster (pStr2); the endothelial cell clusters included main endothelial cells (mEndo) and ribosome-related endothelial cells (rEndo). b–c. Relative abundance of epithelial (b) and stromal (c) clusters within their respective compartments across groups.]d. Comparison of the percentage of proliferative epithelial cells (pEpi, left panel) and proliferative stromal cells (pStr, right panel) between the dynamically cultured EO and HEO complexes under estrogen culture.e. Validation of enhanced proliferation in HEO complexes by flow cytometry. Significantly higher proportions of EdU + cells in epithelial cells of HEO vs. EO complexes under estrogen culture.f. Ligand-receptor pairs exhibiting significant differences in communication probability between HEOe and EOe complexes. Pathways of biological interest include WNT7A, WNT5A , BMP6 , and LGALS9. Dot size represents the maximum communication probability across compared groups; dot color indicates statistical significance ( P < 0.05). Cluster abbreviations: Epi (endometrial epithelial cells), Str (endometrial stromal cells) and Endo (endothelial cells).g. Circle plot showing the BMP6, LGALS9, WNT7A, WNT5A signaling networks among different cell types in EOe and HEOe complexes.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: RNA Sequencing, Cell Culture, Comparison, Biomarker Discovery, Flow Cytometry

Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Endometrial epithelial and stromal cells enhance the formation of vascular networks in HUVECs within the HEO complex through WNT7A and WNT5A signaling pathways. a. Representative images of sprouting assays using HUVEC spheroids treated as indicated. HUVEC spheroids cultured with basic ECM medium are shown as the blank group (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. b. Quantification of sprouting number was determined by Image J software. ∗ P < 0.05 versus Control; # P < 0.05 versus WNT7A; $ P < 0.05 versus DKK1; ^ P < 0.05 versus WNT5A; & P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis. c. Representative images of the tube formation of HUVECs on Matrigel surface treated as indicated. HUVECs cultured with basic ECM medium are shown as the blank (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. d. Quantification of branch number using Image J software. ∗ P < 0.05 versus Control; $ P < 0.05 versus DKK1; &P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis.e. Vascular networks of complex with HUVECs and ESCs (HE), complex with HUVECs and EEOs (HO), and HEO treated with WNT7A (or its inhibitor DKK1) and WNT5A (or its inhibitor BOX5) as indicated. Vascular networks are depicted in green. Scale bar: 100 μm.f. Quantification of the vessel-positive area within the formed vascular network across different groups. ∗P < 0.05, ∗∗P < 0.01, Student's t-test.g. The electrical resistance of HEO and HE with DKK1 and WNT7A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.h. The electrical resistance of HEO and HO with BOX5 and WNT5A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: Protein-Protein interactions, Cell Culture, Control, Positive Control, Software