Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Preclinical evaluation of cysteine protease-inhibitor aloxistatin (E64d) for heart failure therapy

doi: 10.1007/s00109-026-02695-5

Figure Lengend Snippet: Aloxistatin attenuates TGFβ1–induced fibroblast activation and ECM remodeling. A Heatmap of differentially expressed genes (DEGs; adjusted p -value ≤ 0.05, |log 2 fold change|≥ 1) between vehicle (veh) control and transforming growth factor β1 (TGFβ1)-treated HCFs with ICM and DCM backgrounds. B Overrepresentation analysis in DEGs in HCFs after TGFβ1 stimulation using GO, KEGG, Reactome, and WikiPathways databases as references. Selected fibrosis-associated terms are highlighted. C Schematic illustration of experimental design for investigating the effect of aloxistatin on TGFβ1-stimulated HCFs. HCFs were simultaneously stimulated with TGFβ1 and treated with aloxistatin. After 48 h, HCFs were collected for RNA isolation. D Overlap of deregulated genes in HCF after TGFβ1 stimulation and aloxistatin treatment. Values represent the number of genes in each category. E Fold changes in expression of overlapping genes from D in TGFβ1-stimulated HCFs (x-axis) and aloxistatin-treated TGFβ1-stimulated HCFs (y-axis) compared to respective controls. F Relative mRNA expression of fibrosis-associated markers α-smooth muscle actin 2 ( ACTA2 ) and connective tissue growth factor ( CTGF ) after TGFβ1 stimulation and aloxistatin treatment ( n = 3). G Top 30 terms overrepresented in opposite DEGs from E aloxistatin with GO, KEGG, Reactome, and WikiPathways reference databases. H Relative protein level of secreted fibronectin 1 (FN1) and matrix metalloproteinase 2 (MMP2) of TGFβ1-stimulated and aloxistatin treated HCFs. DMSO, dimethyl sulfoxide; ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy

Article Snippet: Pro-fibrotic stimulation was performed with 5 ng/mL TGFβ1 (130–095-067, Miltenyi Biotec, Bergisch Gladbach, Germany) or respective vehicle (veh) control (4 mM HCl containing 0.1% bovine serum albumin; 9048–46-8, Serva, Heidelberg, Germany).

Techniques: Activation Assay, Control, Isolation, Expressing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Preclinical evaluation of cysteine protease-inhibitor aloxistatin (E64d) for heart failure therapy

doi: 10.1007/s00109-026-02695-5

Figure Lengend Snippet: Aloxistatin inhibits inflammatory signaling and lowers oxidative stress. A Venn-diagram showing the overlap of regulated genes in RNA sequencing of rat living myocardial slices (LMS) treated with aloxistatin for 4 h and 24 h compared to respective dimethyl sulfoxide (DMSO) control. B Fold change of inflammation-related candidates significantly downregulated by aloxistatin treatment at both investigated timepoints. C NFκB signaling reported by luciferase activity in human embryonic kidney cells after 24 h treatment with aloxistatin [10 µM, 100 µM] ( n = 3). D Human leukocyte antigen-DR isotype (HLA-DR) expression in interferon γ (IFNγ) [5 µg/mL] stimulated or/and aloxistatin-treated induced pluripotent stem cell-derived macrophages 24 h after treatment ( n = 3). E Reactive oxygen species (ROS) levels assessed by monitoring fluorescence intensity in DCFDA-stained HCF over time after H 2 O 2 stimulation or/and aloxistatin treatment (top). Area under curve (AUC) analysis of ROS levels over time (bottom; n = 3). F Comparison of significant term enrichment (references: GO, KEGG, Reactome, WikiPathways) in overrepresentation analyses from differentially expressed genes in TGFβ1-stimulated HCFs after aloxistatin-treatment (x axis) and LMS cultivated for 24 h under aloxistatin exposure (y axis). RFU, relative fluorescence units

Article Snippet: Pro-fibrotic stimulation was performed with 5 ng/mL TGFβ1 (130–095-067, Miltenyi Biotec, Bergisch Gladbach, Germany) or respective vehicle (veh) control (4 mM HCl containing 0.1% bovine serum albumin; 9048–46-8, Serva, Heidelberg, Germany).

Techniques: RNA Sequencing, Control, Luciferase, Activity Assay, Expressing, Derivative Assay, Fluorescence, Staining, Comparison

Journal: Cell Death Discovery

Article Title: WWP1 gain-of-function drives developmental anoikis through TGFβ pathway during neurodevelopment

doi: 10.1038/s41420-026-02977-4

Figure Lengend Snippet: A Scheme of WWP1 variant-expressing cell line establishment and downstream analysis. The image is created with Biorender.com. B Representative images of dead cell population in WWP1 variants-expressing HeLa cell lines treated with gefitinib. Red, propidium iodide (PI). Scale bar, 200 μm. C Quantification of the PI + counts in HeLa cell lines treated with gefitinib. n = 3 for each condition. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. D Functional GO enrichment analysis of upregulated and downregulated DEGs between WWP1 E798V and control cell lines. The bar color indicates the cluster categories of the GO term. BP, biological process; CC, cellular component. E Differential PROGENy pathway activity scores of WWP1 E798V relative to control. The pathway with the lowest value, TGFβ, is indicated by a deep blue color. F Gene set enrichment plots for TGFβ signaling and epithelial-mesenchymal transition. NES, normalized enrichment score. G Western blot analysis of phospho-SMAD2 and SMAD2 in WWP1 variant-expressing HeLa cell lines treated with vehicle or hTGFβ1 (10 ng/mL). ACTIN is used as the loading control. H Quantification of normalized pSMAD2 to SMAD2 protein expression ratio in WWP1 variant-expressing HeLa cell lines. n = 5. Kruskal-Wallis test with Dunn’s post hoc test. * p < 0.05; ns not significant. Bar graphs indicate mean ± SEM.

Article Snippet: Z-VAD-FMK (20 μM, MedChemExpress, # HY-16658B), recombinant human TGFβ1 (5 μg/mL), or galunisertib (1 or 5 μM, MedChemExpress, #HY-13226) were administered with plasmids intracerebroventricularly during IUE.

Techniques: Variant Assay, Expressing, Functional Assay, Control, Activity Assay, Western Blot

Journal: Cancer Communications

Article Title: Ectopic CD11c Drives SMAD3-Mediated Aberrant Antigen Presentation and Epithelial–Mesenchymal Transition in Esophageal Squamous Cell Carcinoma

doi: 10.34133/cancomm.0014

Figure Lengend Snippet: CD11c mediates phosphorylated SMAD3 nuclear translocation to perform its functions. (A) Left panel, representative confocal images showing p-SMAD3 levels in ITGAX- OE KYSE30 cells treated without or with TGFBR1 inhibitor SB505124. Antibody for each channel is labeled in the panels: p-SMAD3 (green), DiI (red, for cell membrane), and DAPI (blue, for cell nuclei). Right panel, quantitative results of mean fluorescence intensity of p-SMAD3. Data represent mean ± SEM from 3 independent experiments. (B) Left panel, representative confocal images showing ITGAX- KO (sg ITGAX ) KYSE30 cells treated without or with TGFβ1. Antibody for each channel is labeled in the panels: p-SMAD3 (green), DiI (red, for cell membrane), and DAPI (blue, for cell nuclei). Right panel, quantitative results of mean fluorescence intensity of p-SMAD3. Data represent mean ± SEM from 3 independent experiments. (C) Immunoblot of the coimmunoprecipitation products collected from whole cell lysates with SMAD3 (upper panel) or CD11c antibody (lower panel) in KYSE30 cells. (D) Western blot analysis of immunoprecipitation products collected using SMAD3 antibody shows the interaction of TGFBR1-SMAD3 in KYSE30 cells with ITGAX KO or OE. (E) Coimmunoprecipitation assay of the binding of CD11c truncated mutants and SMAD3 (Flag). (F) Cell membrane (marked by Na, K-ATPase), cytoplasm (marked by β-actin), and nucleus (marked by Lamin B1) fractions of ITGAX -OE KYSE30 cells were collected and subjected to IB analysis of p-SMAD3 and SMAD3. (G) Chromatin immunoprecipitation-coupled qPCR assays show enrichment of CD80 or CD86 promoter in cell lysates obtained with anti-p-SMAD3 antibody in KYSE30 cells stimulated with vehicle (Ctrl) or TGFβ1. (H) Luciferase reporter assays in KYSE30 cells using the indicated reporter plasmids of CD80 (left) or CD86 (right) promoters or siRNA targeting SMAD3 . Data are mean ± SEM from 3 experiments, and each had 3 replicates. P values from Student t test. Abbreviations: ITGAX , integrin alpha X; sgCtrl, single guide RNA control; sg ITGAX , single guide RNA targeting ITGAX gene; Vector, control for OE group; OE, overexpression; Δ, deletion; FG-GAP (1 and 2), Phenylalanine-Glycine-GAP repeat fragment 1 to 2; FG-GAP (3 to 7), Phenylalanine-Glycine-GAP repeat fragment 3 to 7; ICD, intracellular domain; Wt- CD80 -P, wild-type CD80 promoter; Mut -CD80 -P, mutant-type CD80 promoter (without p-SMAD3 binding motif); Wt- CD86 -P, wild-type CD86 promoter; Mut- CD86 -P, mutant-type CD86 promoter (without p-SMAD3 binding motif); SMAD3, mothers against decapentaplegic homolog 3; p-SMAD3, phosphorylated SMAD3;. HA, HA tag, CD11c-Δ; Flag, Flag-SMAD3; TGFBR1, transforming growth factor beta receptor I; IB, immunoblot; IP, immunoprecipitation; TGFβ1, transforming growth factor beta 1; Ctrl, control.

Article Snippet: The protein level of TGFβ1 was analyzed using a human TGFβ1 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, Cat. #KE00002) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Labeling, Membrane, Fluorescence, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Control, Plasmid Preparation, Over Expression, Mutagenesis

Journal: Cancer Communications

Article Title: Ectopic CD11c Drives SMAD3-Mediated Aberrant Antigen Presentation and Epithelial–Mesenchymal Transition in Esophageal Squamous Cell Carcinoma

doi: 10.34133/cancomm.0014

Figure Lengend Snippet: Ectopic CD11c expression in epithelial cells is associated with TP53 mutations. (A) Copy number variations of the ITGAX gene in multistage human ESCC development . Each point indicates a micro-biopsy sample. Sample size at each stage: NOR, 603; LGIN, 245; HGIN, 247; and ESCC, 180. (B) Levels of ITGAX copy number (GISTIC score) among human esophageal epithelial clones with different TP53 states . Each point represents one epithelial clone. Sample size for each group: WT, 35; 1 mut, 24; >1 mut, 8; and Loss, 11. (C) Effect of TP53- knockout on ITGAX copy number in human esophageal epithelial cells. Top: Representative images of ITGAX copy number in TP53 -unknockout or TP53 -knockout HET-1A, KYSE150, and KYSE30 cells with ITGAX -specific probe (red, arrow pointed). Bottom: Quantitative statistics of the percentage of cells with ITGAX copy number in TP53 -unknockout versus TP53 -knockout cells. The number of cells ( n ) obtained from 3 independent experiments is indicated on each bar. (D) Immunoblot of CD11c in HET-1A, KYSE150, and KYSE30 cells with or without TP53 KO. Each experiment had 3 biological repeats. (E) Proposed role of ectopically expressed CD11c for cancer cells to escape immune killing and acquire malignant phenotypes. TP53 loss-associated ITGAX amplification results in CD11c ectopic overexpression in epithelial cells. CD11c interacts with SMAD3 and enhances its binding to TGFβ/TGFBR1, promoting SMAD3 phosphorylation. Hyperactivated SMAD3 subsequently translocates to the nucleus, where it suppresses CD80/CD86 transcription while up-regulating the levels of MHC class II molecules. Together, these changes impair tumor cell-mediated antigen presentation and induce EMT, thereby promoting cancer development. In (A) to (C), P values were from the Wilcoxon rank-sum test. ns, not significant. Abbreviations: ITGAX , integrin alpha X; NOR, normal epithelial tissue; INF, inflammatory tissue; LGIN, low-grade intraepithelial neoplasia tissue; HGIN, high-grade intraepithelial neoplasia tissue; ESCC, esophageal squamous cell carcinoma; GISTIC, Genomic Identification of Significant Targets in Cancer; WT, wild type; 1 mut, one mutation; >1 mut, multiple mutations; Loss, TP53 biallelic loss; Ctrl, control; EMT, epithelial–mesenchymal transition; SMAD3, mothers against decapentaplegic homolog 3; P, phosphorylation; TGFβ, transforming growth factor beta; TGFBR1, transforming growth factor beta receptor I; Treg, regulatory T cell; MHC II, major histocompatibility complex class II.

Article Snippet: The protein level of TGFβ1 was analyzed using a human TGFβ1 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, Cat. #KE00002) according to the manufacturer’s instructions.

Techniques: Expressing, Clone Assay, Knock-Out, Western Blot, Amplification, Over Expression, Binding Assay, Phospho-proteomics, Immunopeptidomics, Mutagenesis, Control