Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) Confocal imaging of ITGα2, Vimentin, and K8 in an IDC patient tissue section. Green contour: cluster of cancer cells with protrusive morphology; green arrowheads: basal-like cells (K8-low) at the tumor–stroma interface with high ITGα2 expression. White contour: cluster of cancer cells lacking basal-like cells (K8-high), with low ITGα2 expression; magenta arrowheads. White arrowheads: fibroblast-like cells (elongated, spindle-shaped). (B) Quantification of mean gray values for ITGα2 and Vimentin in basal-like (n = 33), luminal-like (n = 32), and fibroblast-like (n = 32) cells from one IDC patient tissue section. (C) Representative brightfield images of MMTV-PyMT organoids (ITGα2-WT or ITGα2-KO, gRNA1 and gRNA2) cultured in 3D Collagen I. Black arrowheads: invasive strands. (D) Percentage of organoids exhibiting one or more invasive strands in ITGα2-WT and ITGα2-KO (clones 1 and 2 from gRNA1) MMTV-PyMT organoids. (E) qPCR analysis of classical TGF-β and EMT target genes in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Values represent mean normalized mRNA expression (relative to housekeeping genes), shown for KO organoids relative to WT controls (dashed line). Data are presented as mean ± SD from three independent experiments. (F) Confocal imaging of Col ¾ and F-actin in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids after one day in 3D Collagen I. (G, H) qPCR analysis of Vimentin and Slug mRNA expression in ITGα2-KO versus ITGα2-WT MMTV-PyMT organoids treated with Activin A (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graphs represent mean normalized expression values ± SD from four independent experiments. (I) Kaplan–Meier analysis correlating high vs. low mRNA expression of INHBA, ITGA2, ITGB1, and their combinations (ITGA2 + ITGB1, or INHBA + ITGA2 + ITGB1) with distant metastasis-free survival (DMFS) in patients with grade 3 breast cancer. Scale bars: 100 μm (A, C), 50 μm (A, zoom-in), 50 μm (F), 10 μm (F, zoom-in). P values: two-sided unpaired Mann–Whitney test (E), two-sided Kruskal-Wallis test with Dunn’s multiple comparisons (G, H), Log-rank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Imaging, Expressing, Cell Culture, Clone Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) DNA sequence of the Itgα2 gene to confirm gene knockout by Crispr-Cas9 gene editing. Red regions indicate the insertion of one base pair compared to the wildtype Itgα2 sequence. (B) Western blot analysis showing Itgα2 and GAPDH expression from whole cell lysates of MMTV-PyMT organoids WT or KO (gRNA1 or gRNA2). (C) Confocal imaging of Itgα2 and K8 in MMTV-PyMT organoids with Itgα2 WT or knockout (KO) grown in Collagen I for 3 days. White arrowheads: invading strands in Itgα2 WT organoids led by basal-like cells (low K8, high Itgα2, insets), White arrows: non-invading basal-like cells at the ECM interface in Itgα2 KO organoids (low K8, low Itgα2, insets). (D) Percentage of invasive organoids in MMTV-PyMT organoids Itgα2-WT versus Itgα2-KO (gRNA2). (E) Mean gray value of Col ¾ relative to Collagen I reflection in MMTV-PyMT organoids with Itgα2-WT and Itgα2-KO. Median: red lines, from n = 11 organoids per group from two independent experiments. (F) Single confocal slice showing INHBA and K8 expression in Itgα2-WT and Itgα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Insets show basal-like cells (K8 low) guiding invasive strands (Itgα2-WT) or remaining at the organoid rim (Itgα2 KO, non-invasive). (G) Mean-gray value of Inhba in basal-like cells (K8-low) located at the rim of Itgα2 WT vs. KO MMTV-PyMT organoids (3D Collagen I, day 1). Median: red lines, n =11 cells from 6 Itgα2-WT organoids, and n = 12 cells from 7 Itgα2-KO organoids from one experiment. (H) qPCR analysis showing relative mRNA expression of CTGF in MMTV-PyMT Itgα2-KO organoids compared to Itgα2-WT organoids treated with Activin A ligand (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graph represents mean normalized expression values (relative to housekeeping genes) ± SD from four independent experiments. (I) Kaplan-Meier plot correlating ITGB1 gene expression with DMFS in grade 3 breast cancer patients. Scale bars: 50 μm (C, F), 25 μm (C, F, Zoom in). P values, two-sided unpaired Mann–Whitney test (E, G), two-sided Kruskal-Wallis test (Dunn’s multiple comparison) (H), Logrank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Sequencing, Gene Knockout, CRISPR, Western Blot, Expressing, Imaging, Knock-Out, Cell Culture, Control, Gene Expression, MANN-WHITNEY, Comparison

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Activin A and Follistatin serum concentrations in patients with thymic epithelial tumors and Myasthenia Gravis Patients without TETs. Plots show preoperative Activin A and Follistatin serum concentrations in patients with thymic carcinomas (TCs), thymomas and healthy volunteers ( A , B ), and in patients with TETs according to Masaoka - Koga tumor stage ( C , D ). Further, the impact of tumor resection on postoperative Activin A and Follistatin serum levels is indicated ( E , F ). In addition, analysis of Activin A and Follistatin serum concentrations are illustrated in myasthenic patients (MG) compared to volunteers ( G,H ). Scatter dot plots are indicated as mean ± standard error of the mean.

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques:

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Activin A and Follistatin serum concentrations in patients with thymic epithelial tumors.

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques:

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Activin A and Follistatin expression in thymic malignancies. Strong cytoplasmic and membranous Activin A expression was found in all TETs. Representative Activin A staining is shown in a WHO type AB ( A ), B2 ( C ), and B3 ( E ) thymoma. Conversely, nuclear and cytoplasmic Follistatin staining was found in MNT ( B ), B2 thymoma ( D ), and TC ( F ) (magnification × 400; scale bar: 40 μm). WHO , World Health Organization ; TC , thymic carcinoma ; MNT , micronodular thymoma .

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Microvessel Density in thymic epithelial tumors. CD34 staining of a Masaoka - Koga stage I WHO type B2 thymoma ( A ), a stage II A thymoma ( B ) and a stage III A thymoma ( C ) are shown (200 × magnification; scale bar: 80 μm). Preoperative Follistatin serum concentrations showed a significant negative correlation ( D ) with the number of immature tumor vessels (r: −0.443; p = 0.014), and a significantly positive correlation ( E ) with the amount of mature tumor vessels (r: 0.549; p = 0.002). While the amount of immature tumor vessels decreased with higher tumor stage ( F ), the number of mature tumor vessels increased with level of invasiveness ( G ). Similarly, total numbers of tumor vessels decreased from stage I to stage IV tumors ( H ). High preoperative Follistatin serum levels were associated with significantly worse FFR ( I ).

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques: Staining

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Spearman correlation was performed to analyze relationships between microvessel density and different systemic inflammatory markers.

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques:

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Survival Analyses.

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques:

Journal: Scientific Reports

Article Title: Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors

doi: 10.1038/s41598-019-53671-8

Figure Lengend Snippet: Univariable and Multivariable Cox-Regression Analyses.

Article Snippet: We used monoclonal rabbit anti-human Anti-Activin A Receptor Type IB antibody (ab204655, Abcam, Cambridge, UK) and monoclonal mouse anti-human Follistatin antibody IgG 2a (MAB669, R&D Systems, Minneapolis, MN, USA) as primary antibodies to assess Activin A and Follistatin expression in TETs.

Techniques:

Journal: Biology of reproduction

Article Title: Transforming growth factor beta1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling.

doi: 10.1093/biolre/iox140

Figure Lengend Snippet: Figure 6. Analyzing the fibrotic effects of TGF-β1 on EdSCs in vitro. After EdSCs were exposed with TGF-β1, fibrosis-related genes including COL1A1, ACTA2, CTGF, and FN1 at 4, 12, 24, 48, and 72 h were detected by RT-qPCR (A), and fibrosis-related markers including COL1A1, ACTA2, CTGF, and MMP2 were analyzed by western blotting (B). The components’ expression of the TGF-β1/Smad signaling pathway such as SMAD2, p-SMAD2, SMAD3, and p-SMAD3 were demonstrated by western blotting analysis (C).

Article Snippet: Briefly, a series of 5-μm sections was incubated with the following antibodies: monoclonal rabbit antibodies against human TGFB1 (1:200), TGF-β1 receptor (TGFBR1; 1:500; sc-398; Santa Cruz Biotechnology), SMAD2 (1:200), SMAD3 (1:200), SMAD4 (1:200), SMAD2/3 (1:200), phospho-SMAD2 (p-SMAD2; 1:200), phospho-SMAD3 (p-SMAD3; 1:200), Collagen I (COL1A1, 1:200), connective tissue growth factor (CTGF, 1:1000), alpha-smooth muscle actin (ACTA2, 1:200), cluster of differentiation 10 (CD10, 1:200; ab126593; Abcam), and a monoclonal mouse antibody against human pan cytokeratin (KRT (pan); 1:200; ab7753; Abcam).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Expressing

Journal: Biology of reproduction

Article Title: Transforming growth factor beta1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling.

doi: 10.1093/biolre/iox140

Figure Lengend Snippet: Figure 7. IHC detection of activated TGF-β1/Smad signaling in the cystic walls of endometriomas. (A) Representative photomicrograph of an excised endometri- oma stained with Masson’s trichrome after laparoscopic cystectomy. The red lines separated ovarian tissue and the cystic wall, while yellow lines separated the endometriotic loci and the cystic wall. Follicles were highlighted with arrows in (A) and identify ovarian tissue. The expression of TGFB1 (B) and TGFBR1 (C), and fibrosis-related factors including ACTA2 (D), COL1A1 (E), and CTGF (F), and components of the TGF-β1/Smad signaling pathway including p-SMAD2 (G), p-SMAD3 (H), SMAD2/3 (I) SMAD2 (J), SMAD3 (K), and SMAD4 (L) were demonstrated in the serial sections. (M) A comparison among the percentage of positive expression areas of different primary antibody in the cystic walls of endometriomas. Magnified photomicrographs with high-resolution view areas of the blue-square frames were shown in Supplemental Figure S4 correspondingly. (A–L: 100×).

Article Snippet: Briefly, a series of 5-μm sections was incubated with the following antibodies: monoclonal rabbit antibodies against human TGFB1 (1:200), TGF-β1 receptor (TGFBR1; 1:500; sc-398; Santa Cruz Biotechnology), SMAD2 (1:200), SMAD3 (1:200), SMAD4 (1:200), SMAD2/3 (1:200), phospho-SMAD2 (p-SMAD2; 1:200), phospho-SMAD3 (p-SMAD3; 1:200), Collagen I (COL1A1, 1:200), connective tissue growth factor (CTGF, 1:1000), alpha-smooth muscle actin (ACTA2, 1:200), cluster of differentiation 10 (CD10, 1:200; ab126593; Abcam), and a monoclonal mouse antibody against human pan cytokeratin (KRT (pan); 1:200; ab7753; Abcam).

Techniques: Staining, Expressing, Comparison

Journal: Journal of Immunology Research

Article Title: Hair Follicle Dermal Sheath Derived Cells Improve Islet Allograft Survival without Systemic Immunosuppression

doi: 10.1155/2015/607328

Figure Lengend Snippet: DSCC stimulates reduced alloimmune responses and promotes beta cell survival in vitro . (a) Immune privilege related genes were expressed in DSCC. Hair follicle dermal sheath cup cells (DSCC), dermal papilla (DP), and dermal sheath (DS) cell gene expression were examined relative to nonfollicular fibroblasts (FB). Values were obtained using the 2 −ΔΔCT fold change method. For standard error (SE), the standard deviation of relevant samples was divided by the square root of the number of samples. The calculation for error bars was 2 −ΔΔCT + SE minus fold change or fold change minus 2 −ΔΔCT − SE to find the range of difference. The fold change was generated from duplicated values ( n = 3). (b) DSCC significantly inhibited allogeneic leukocyte IFN γ secretion relative to FB. DSCC or FB from B6 (2 × 10 4 ) were cocultured with splenic leukocytes (Balb/c, 2 × 10 5 ) for 5 days. IFN γ detected in supernatant was significantly less in the presence of DSCC than FB. Data are shown as mean pg/mL IFN γ of three different experiments ± SEM, with Student t -test showing significance, ∗ P < 0.05. Percentage of IFN γ expressing cells in CD4 + or CD8 + subsets after 5 days of coculture was presented by flow cytometric analysis. Representative dot plots and accumulated data from CD4 + IFN γ + or CD8 + IFN γ + cell populations are shown. (c) DSCC preserved beta cell function. DSCC or FB from B6 mice were cocultured with islets from Balb/c mice; the ability of insulin secretion was measured by static incubation at 3 and 7 days and relative gene expression was detected by qPCR at day 7. A similar insulin secretion upon glucose stimulation was detected at day 3. However, better insulin secretion was observed at day 7 with 20 mM glucose stimulation. Increased insulin mRNA and decreased Fas and Bax mRNA gene expression were detected at culture day 7. BMP6: bone morphogenetic protein 6; Fgf2: fibroblast growth factor 2; Il1ra: interleukin 1 receptor antagonist; Inhba: Inhibin beta A; Tap2: transporter associated with antigen processing. Data represent 3 independent experiments and are expressed as means ± SEM. Statistical significance was determined by Student t -test, ∗ P < 0.05.

Article Snippet: The membrane was blocked in PBS containing 5% skim milk for 1 h followed by incubation with the primary antibody rabbit anti-mouse BMP6 (ab15640), Inhibin beta A (22689-1-AP), Fgf2 (sc-79, Santa Cruz, Dallas, TX), or mouse anti-actin.

Techniques: In Vitro, Gene Expression, Standard Deviation, Generated, Expressing, Cell Function Assay, Incubation