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: Molecular Medicine Reports

Article Title: Transplantation of ovarian granulosa-like cells derived from human induced pluripotent stem cells for the treatment of murine premature ovarian failure

doi: 10.3892/mmr.2016.5191

Figure Lengend Snippet: Primary antibodies, their source and dilutions.

Article Snippet: Rabbit anti-human/mouse inhibin α (sc-30146) , Santa Cruz Biotechnology, Inc. , 1:100.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Transplantation of ovarian granulosa-like cells derived from human induced pluripotent stem cells for the treatment of murine premature ovarian failure

doi: 10.3892/mmr.2016.5191

Figure Lengend Snippet: Immunofluorescence staining. Immunohistochemical staining indicated that ovarian granulosa-like cells derived from induced pluripotent stem cells strongly expressed ovarian granulosa cells markers [anti-Müllerian hormone (AMH), inhibin α, inhibin β and follicle-stimulating hormone receptor (FSHR)], but not stem cell markers [octamer-binding transcription factor 4 (Oct4), SRY (sex-determining region Y)-box 2 (Sox2), Nanog and stage-specific embryonic antigen-4 (SSEA4) 12 days post-induction. Each sample was stained for two markers with the color of the fluorescence indicated by the color of the word. Original magnification, ×200. Cy3, cyanine 3; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Article Snippet: Rabbit anti-human/mouse inhibin α (sc-30146) , Santa Cruz Biotechnology, Inc. , 1:100.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Derivative Assay, Binding Assay, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Transplantation of ovarian granulosa-like cells derived from human induced pluripotent stem cells for the treatment of murine premature ovarian failure

doi: 10.3892/mmr.2016.5191

Figure Lengend Snippet: Primary antibodies, their source and dilutions.

Article Snippet: Rabbit anti-human/mouse inhibin β (sc-50288) , Santa Cruz Biotechnology, Inc. , 1:100.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Transplantation of ovarian granulosa-like cells derived from human induced pluripotent stem cells for the treatment of murine premature ovarian failure

doi: 10.3892/mmr.2016.5191

Figure Lengend Snippet: Immunofluorescence staining. Immunohistochemical staining indicated that ovarian granulosa-like cells derived from induced pluripotent stem cells strongly expressed ovarian granulosa cells markers [anti-Müllerian hormone (AMH), inhibin α, inhibin β and follicle-stimulating hormone receptor (FSHR)], but not stem cell markers [octamer-binding transcription factor 4 (Oct4), SRY (sex-determining region Y)-box 2 (Sox2), Nanog and stage-specific embryonic antigen-4 (SSEA4) 12 days post-induction. Each sample was stained for two markers with the color of the fluorescence indicated by the color of the word. Original magnification, ×200. Cy3, cyanine 3; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Article Snippet: Rabbit anti-human/mouse inhibin β (sc-50288) , Santa Cruz Biotechnology, Inc. , 1:100.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Derivative Assay, Binding Assay, Fluorescence