Journal: Life Science Alliance

Article Title: Variants of Epas1 contribute to hypoxia adaptation in the subterranean rodents Eospalax and Spalax

doi: 10.26508/lsa.202603622

Figure Lengend Snippet: (A) mRNA levels of Epas1 and its target gene Cited2 were significantly higher in E. baileyi LFs compared with rats under normoxia, whereas Col3a1 was much lower in E. baileyi LFs. (B) Hypoxia up-regulated the mRNA expression of Col3a1 in rat LFs, but only induced Epas1 gene expression in E. baileyi LFs. (C) TGF-β treatment significantly induced an increase in Col3a1 mRNA levels in rat LFs, but in E. baileyi LFs, Epas1 and Cited2 mRNA levels significantly increased, whereas Col3a1 mRNA showed no significant change. (D) Western blot analysis revealed that hypoxia promoted COL1A1 protein expression in rat cells, whereas hypoxia inhibited COL1A1 protein expression in E. baileyi LFs. (E) mRNA expressions of collagen family genes and interferon-related genes were decreased in E. baileyi LFs under hypoxia. (F) COL1A1 protein showed much lower fluorescence intensity in E. baileyi LFs compared with rat cells in immunofluorescence analysis. (G, H) TGF-β treatment significantly induced fibrosis in primary rat LFs, but had no significant effect on E. baileyi LFs. Scale bar: 30 μm. Data are the mean ± SEM, with each point representing one replicate mean. Statistical comparisons among different treatments were performed using a one-way ANOVA and paired t test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus rat LF or control, n = 3.

Article Snippet: Cells were moved to the hypoxia incubator in which the O 2 level was 0.1% for 24 h. Cells were treated with 10 μM Raf kinase inhibitor LY3009120, 10 μM ATM kinase inhibitor KU-55933, 20 μM PKA inhibitor H89 2HCL, 10 μg/ml specific inhibitor of AKT MK-2206 2HCL (Selleck), or 10 μM EPAS1 inhibitor belzutifan PT2977 (MCE) for 24 h, 5 ng/ml TGF-β (MCE) for 48 h, and 1 M H 2 O 2 for 6 h before qRT-PCR, Western blot, and immunofluorescence analyses.

Techniques: Expressing, Gene Expression, Western Blot, Fluorescence, Immunofluorescence, Control

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: (A) Schematic of the multilayer perceptron with eight input nodes (BMP4, CD248, GRP, LGR4, MGP, TGFB3, ERBB3, ETS2), a five-node hidden layer and a two-node output layer encoding Control (normal) and Treatment (GC). (B) Receiver operating characteristic (ROC) curve and area under the curve (AUC) in the training cohort (TCGA-STAD). (C) ROC curve and AUC in the independent validation cohort ( GSE54129 ).

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Control, Biomarker Discovery

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: Kaplan–Meier analysis of OS in gastric cancer patients stratified by mRNA expression of network-prioritized genes: (A) BMP4, (B) CD248, (C) GRP, (D) LGR4, (E) MGP, (F) TGFB3, (G) ERBB3 and (H) ETS2.

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: Multiplex immunofluorescence validation of hub gene expression in the gastric cancer tumor microenvironment. (A) Representative staining of BMP4 (red) and Pan-CK (green) in gastric cancer showing predominant localization of BMP4 to Pan-CK + tumor epithelial nests. (B) CD248 (red) colocalizes with FAP + cancer-associated fibroblasts (CAF; green) within the stromal compartment. (C) TGFB3 (red) is enriched in the FAP + CAF-rich stroma. (D) LGR4 (red) is detected both in FAP + CAFs (green) and in Pan-CK + tumor epithelial cells (yellow). (E) MGP (red) is present in CD31 + endothelial structures (yellow) and in FAP + CAF-rich stromal regions (green). Nuclei are counterstained with DAPI (blue). Left panels show merged images; right panels display the corresponding single-channel views. Scale bars, 100 μm. Integrating the proteome-wide MR screen with PPI topology, survival analysis, and compartment-resolved expression patterns, we prioritized ERBB3, LGR4, BMP4, CD248, MGP, TGFB3, GRP, and ETS2 as network-prioritized candidates for downstream contextualization. Among them, TGFB3 was selected for focused computational and cellular characterization on the basis of several convergent prioritization features, including its nominal MR association, network-topology ranking, association with overall survival, fibroblast-enriched expression pattern, and spatial organization in gastric cancer tissues. These observations provided a rationale for additional investigation. Accordingly, subsequent virtual screening, molecular docking, molecular dynamics simulation, and recombinant-TGFB3 perturbation experiments were performed as exploratory analyses to evaluate structural plausibility and cellular responses associated with TGFB3 exposure.

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Multiplex Assay, Immunofluorescence, Biomarker Discovery, Gene Expression, Staining, Expressing, Recombinant

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: Molecular docking of TGFB3 with candidate ligands. The left panel in each subfigure depicts the three-dimensional ligand orientation within the TGFB3 binding pocket; the right panel presents a two-dimensional interaction diagram, highlighting hydrogen bonds and hydrophobic contacts. (A) Docking pose of TGFB3 with proflavine hemisulfate. (B) Docking pose of TGFB3 with hydroxychloroquine sulfate. (C) Docking pose of TGFB3 with rizatriptan benzoate. (D) Docking pose of TGFB3 with L-histidine. (E) Docking pose of TGFB3 with retigabine. (F) Heatmap of binding affinities (kcal/mol) obtained from molecular docking analyses between TGFB3 and the five ligands (proflavine hemisulfate, hydroxychloroquine sulfate, rizatriptan benzoate, L-histidine and retigabine).

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Binding Assay

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: Molecular dynamics analysis of the TGFB3–proflavine hemisulfate complex over 100 ns. (A) Temporal evolution of the RMSD of the protein–ligand complex. (B) Time-dependent changes in the overall Rg and its axis-specific components (Rg_x, Rg_y, Rg_z). (C) SASA as a function of simulation time. (D) Two-dimensional Gibbs free energy landscape derived from principal component analysis (PC1–PC2), with the color gradient indicating free energy magnitude (kJ/mol). (E) Covariance matrix heatmap of atomic motions, where color intensity reflects the strength and direction (positive or negative) of residue–residue correlations. (F) Three-dimensional Gibbs free energy surface and its projected contour map on the PC1/PC2 plane, visually depicting the energy distribution along the principal components.

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Derivative Assay, Residue

Journal: Frontiers in Oncology

Article Title: From proteome-wide Mendelian randomization and multi-omics integration to functional validation: TGFB3 as a prioritized candidate in gastric adenocarcinoma

doi: 10.3389/fonc.2026.1883227

Figure Lengend Snippet: (A) NicheNet ligand–receptor analysis using fibroblasts as sender cells and epithelial cells as receiver cells, with TGFB3 specified as the ligand of interest. Candidate receptors associated with TGFB3 are displayed. (B) Scatter plot showing the relationship between TGFB3 and TGFBR2 expression. The Spearman correlation coefficient and corresponding P value are indicated in the panel. (C) Manifold-alignment plot generated after in silico knockout of TGFBR2 in epithelial cells using scTenifoldKnk. Each point represents one gene and selected genes are labeled. (D) Ranking of genes affected following virtual TGFBR2 knockout according to the perturbation statistics generated by scTenifoldKnk. (E) Functional enrichment analysis result of genes affected by virtual TGFBR2 knockout.

Article Snippet: Proflavine hemisulfate (purity > 98%, HY-B0883) and recombinant human TGFB3 (rhTGFB3; purity > 95% by reducing SDS–PAGE, HY-P700152AF) were purchased from MedChemExpress (NJ, USA).

Techniques: Expressing, Generated, In Silico, Knock-Out, Labeling, Functional Assay

Journal: bioRxiv

Article Title: Fitness and immune-escape within germinal centers shape premalignant evolution toward lymphoma

doi: 10.64898/2026.06.25.734549

Figure Lengend Snippet: (A) Experimental design for single-cell RNA sequencing of PD-1⁺ CD8⁺ T-cells sorted from recipient mice 10 days after immunization. (B) UMAP projection of PD-1⁺ CD8⁺ T-cells colored by annotated subset identity. (C) Dot plot showing expression of selected marker genes across annotated CD8⁺ T-cell subsets. (D) Density plots showing the distribution of PD-1⁺CD8⁺ T-cells from Ctrl, BC, and BCK recipients across the UMAP. (E) UMAP projections showing expression of selected marker genes from (C). (F) Slingshot trajectory analysis of PD-1⁺ CD8⁺ T-cell differentiation, highlighting trajectory path1 from activated states toward stem-like, early effector and effector states. (G) Comparison of CD8⁺ T-cell subset distribution along path 1, which terminates in cytotoxic effector differentiation. Quantification shows mean cluster proportion from two biological replicates. (H) Gene set enrichment analysis of HALLMARK pathways comparing cluster 4 from BCK and BC conditions. (I-K) TGF-β receptor blockade following GC response derived from transferred BCK B-cells. (I) Experimental design for LY2109761 treatment. (J) Left, representative gating of granzymeB⁺ perforin⁺ effector cells among PD-1⁺ CD8⁺ T-cells. Right, quantification of PD-1⁺ CD8⁺ T-cells and granzymeB⁺ perforin⁺ effector CD8⁺ T-cells per 10⁶ splenocytes. (K) Left, representative gating of donor-derived reporter⁺ CD45.2⁺ B-cells. Right, quantification of donor-derived reporter⁺ CD45.2⁺ GC B-cells per 10⁶ splenocytes. (L) Endogenous CD45.1⁺ GC B-cell response to SRBCs in the absence or presence of TGF-β receptor inhibition with LY2109761. Left representative gating of host CD45.1⁺ GC B-cell B-cells. Right, quantification of host CD45.1⁺ GC B-cell B-cells. (M) Requirement for CD8⁺ T-cells during TGF-β receptor blockade. Top, schematic of experimental design. Bottom, representative gating of donor-derived reporter⁺ CD45.2⁺ B-cells from BCK recipients treated with LY2109761 together with isotype control or anti-CD8 antibody; and quantification of reporter⁺ CD45.2⁺ GC B-cells per 10⁶ splenocytes. Each symbol in (J, L, K, M) represents an individual mouse. Each symbol in (G) represents mean value from two biological replicates. (J-L) placebo n = 3, LY2109761 n = 3. (M) LY2109761 + isotype n = 3, LY2109761 + anti-CD8 n = 3. Small horizontal lines indicate mean ± SD. Data in (J-L) are representative of two independent experiments; data in (K) are representative of two independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; Two-way ANOVA in (G) , value calculated on two biological replicates from each group, remaining statistical calculations used unpaired two-tailed Student’s t test. ns, not significant.

Article Snippet: For TGF-β pathway inhibition, mice were treated with the dual TGF-β type I/II receptor inhibitor LY2109761 (MedChemExpress, HY-12075; 40 mg/kg).

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Cell Differentiation, Comparison, Derivative Assay, Inhibition, Control, Two Tailed Test