Journal: bioRxiv

Article Title: Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission

doi: 10.1101/2025.09.15.676370

Figure Lengend Snippet: Fluorescein permeability assay of epithelial cell cultures in ALI at 0, 3, 9, and 12 days.

Article Snippet: Cells from the immortalized human epithelial cell line NuLi-1 (from ATCC) were expanded in a T25 flask for 4–5 days in serum-free Airway Epithelial Cell Basal Medium (BEGM; ATCC PCS-300-030) with additives from the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) as described .

Techniques: Permeability

Journal: bioRxiv

Article Title: Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission

doi: 10.1101/2025.09.15.676370

Figure Lengend Snippet: Confocal microscopy images of NuLi-1 lung epithelial cells in the ALI model after 12 days, stained for F-actin and with DAPI to highlight nuclei.

Article Snippet: Cells from the immortalized human epithelial cell line NuLi-1 (from ATCC) were expanded in a T25 flask for 4–5 days in serum-free Airway Epithelial Cell Basal Medium (BEGM; ATCC PCS-300-030) with additives from the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) as described .

Techniques: Confocal Microscopy, Staining

Journal: Scientific Reports

Article Title: TGFβ3-mediated induction of Periostin facilitates head and neck cancer growth and is associated with metastasis

doi: 10.1038/srep20587

Figure Lengend Snippet: ( A,B ) The transcriptional and translational statuses of POSTN were determined in CAFs and NFs, 6 representative HNC cell lines and normal oral epithelial cells (titled normal) using western blotting ( A ) and real-time PCR ( B ). ( C ) Representative images of immunohistochemical staining showed the distribution of POSTN expression in adjacent normal tissues and HNC tissues. ( D,E ) POSTN expressions in NFs and CAFs were detected using immunohistochemical staining ( D ) and confocal microscopy ( E ). ( F ) The protein levels of POSTN, FAP and α-SMA were determined by a western blot analysis. ( G ) POSTN mRNA levels in 5 pairs of NFs and CAFs were measured by real-time PCR. ( H ) POSTN protein levels in the media of NFs and CAFs were determined by ELISA analyses. (**** p < 0.0001).

Article Snippet: The blots were probed with a mouse monoclonal POSTN antibody (Sigma, USA, 1:1 000), a rabbit polyclonal POSTN antibody [polyclonal] (Sigma, USA; 1:1 000), a mouse monoclonal α-SMA antibody [1A4] (Abcam, USA; 1:300) and a rabbit monoclonal FAP antibody [S9B] (Abcam, USA; 1:1 000).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: The Shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets

doi: 10.1002/dvdy.21942

Figure Lengend Snippet: (A) Schematic depicting the sections of embryo imaged for B through G. (B) Lateral line placode, (C) Proctodeum, (D) Cement gland (phalloidin) and (E) Otic vesicle. green is α-tubulin, red is propidium iodide in panel B, C, E. green is phalloidin in panel D. (F–G) Fluorescence images of in situ hybridization against Shroom3, DIC: differential interference contrast image, α-tubulin: image stained with α-tubulin antibody, NBT/BCIP: NBT/BCIT precipitate following in situ hybridization by Shroom3 probe. It was detected by confocal microscopy (F) otic vesicle (G) lateral line placode. Scale bar = 50 µm.

Article Snippet: Monoclonal anti-α-tubulin antibody (1:300 dilution, DM1A, Sigma) and rabbit polyclonal anti-γ-tubulin antibodies (1:300 dilution, Abcam) were used for primary antibodies and Alex Fluor-488 goat anti-mouse or anti-rabbit IgG was used for secondary antibody (Invitrogen, 1:300 dilution).

Techniques: Fluorescence, In Situ Hybridization, Staining, Confocal Microscopy

Journal:

Article Title: The Shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets

doi: 10.1002/dvdy.21942

Figure Lengend Snippet: (A–C) Ectopic Shroom2 induces cell heightening in naïve epithelial cells. (A) Control (B) Shroom2 expressing cell (green: γ-tubulin, red: myc-Shroom2) (C) Graph of apicobasal cell height of control and Shroom2 expressing cells (mean ± s.e.m; ctl, n=16;Shroom2, n=15). (D–H) Ectopic Shroom2 expression induces cell heightening in epidermal cells. (D) Control, (E) Shroom2 expressing and (F) Shroom3 expressing epidermis (green: α-tubulin, red: myc-tagged Shroom2 or 3). (G) Graph of apicobasal cell height in Shroom2 expressing epidermal cells (mean ± s.e.m: ctl, n=25;Shroom2, n=25). (H) Graph of apicobasal cell height in Shroom3 expressing epidermal cells (mean ± s.e.m: ctl, n=39;Shroom3, n=40). Scale bar = 20 µm. Yellow arrow bars are showing the way to measure the cell height for control cells (neighboring cells) and pink arrow bars are showing that for Shroom expressing cells. (I–L) Loss of function assay for Shroom2 in neural plate. Transverse cross-section view of neural plate (green: α-tubulin, red: propidium iodide). (I) One side-Shroom2 mismatched morpholino (MM) injected embryo. (J) One side-Shroom2 morpholino (MO) injected embryo. The high magnification views of control side cells (K) and Shroom2 MO injected side cells (L). Scale bar = 50 µm.

Article Snippet: Monoclonal anti-α-tubulin antibody (1:300 dilution, DM1A, Sigma) and rabbit polyclonal anti-γ-tubulin antibodies (1:300 dilution, Abcam) were used for primary antibodies and Alex Fluor-488 goat anti-mouse or anti-rabbit IgG was used for secondary antibody (Invitrogen, 1:300 dilution).

Techniques: Control, Expressing, Functional Assay, Injection

Journal: bioRxiv

Article Title: VE-cadherin endocytosis controls vascular integrity and patterning during development

doi: 10.1101/769182

Figure Lengend Snippet: A , Amino acid sequence of wild type VE-cad core p120-binding domain and mutants analyzed in this study. The wild type allele binds p120 and undergoes endocytosis upon p120 dissociation. The VE-cad GGG allele contains alanine substitutions of GGG residues (purple box), which disrupt p120 binding leading to increased endocytosis. The VE-cad DEE endocytic mutant, with mutated DEE residues (blue box), exhibits partially reduced p120 binding, yet fails to undergo endocytosis. The ΔJMD endocytic mutant contains an 11 amino deletion comprising the DEE signal and surround residues, and also fails to bind p120 or undergo endocytosis. B , Schematic representations of the VE-cad mutants in part A. C , Sanger sequencing chromatograms of the indicated VE-cadherin homozygous mutant mice.

Article Snippet: Primary antibodies were: rat anti-VE-cad (BV13, eBioscience # 14144181, 1:500); rabbit anti-p120 (S-19, Santa Cruz Biotechnology, Inc #sc-1101, 1:250); mouse anti-beta-catenin (BD Biosciences #610153, 1:250); mouse anti-N-cadherin (BD Biosciences #610920, 1:250).

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: VE-cadherin endocytosis controls vascular integrity and patterning during development

doi: 10.1101/769182

Figure Lengend Snippet: A , Genotyping analysis of postnatal offspring from VE-cad GGG/+ intercrosses reveals less than expected number of VE-cad GGG/GGG homozygous mutants, indicating partial lethality. Genotyping was performed between P6-P8 and the expected number of mice was based on the total number of mice and expected Mendelian ratios. ***p<0.001 in Χ 2 analysis. B , Image of male VE-cad +/+ and VE-cad GGG/GGG littermates at 6 weeks of age illustrating small size of VE-cad GGG/GGG mutants. C , Decreased body weight in VE-cad GGG mutant mice at 3 weeks and 6 weeks. Body weight was assessed in VE-cad +/+ and VE-cad GGG/GGG males and females. Results are shown as mean ± SEM. *p<0.05, **p<0.001. D , Left and middle panels: Gross examination of VE-cad +/+ (top) and VE-cad GGG/GGG mutant (bottom) whole embryos at E12.5. Variable size hemorrhages (arrows) were observed in the VE-cad GGG/GGG embryos, which are shown at larger magnification in the middle panels. Right panels: Fixed eye cups from VE-cad +/+ and VE-cad GGG/GGG mutant mice at P3. Larger and more frequent hemorrhages (arrows) were observed in the retinas of VE-cad GGG/GGG mutants compared to littermates. Scale bar (left and middle): 1 mm. Scale bar (right): 0.3 mm. E , Increased vascular permeability in VE-cad GGG/GGG mutant mice. Lung permeability in three month old mice in response to LPS treatment was assessed by the Evans Blue dye method 6 hours after treatment. The lungs were harvested and dye extravasation was quantified spectrophotometrically and normalized to lung dry weight. The bar graph represents means ± SEM with 6-7 mice per group. Two-way ANOVA *p<0.0437, ***p<0.0001, **p<0.0037. F , Visualization of the retinal vasculature by Isolectin-B4 staining at P3 revealed normal blood vessel patterning in VE-cad GGG/GGG mutant retinas (right) compared to VE-cad +/+ (left) littermates. Panels on the right show higher magnification of the boxed region at the vessel front in left panels. Scale bar: 300 μm. G , Quantitation of vascular parameters at the vessel front in VE-cad GGG/GGG mutant retinas at P3. Data are presented as % of WT littermate control and represent mean ±S.E.M, n=5 independent litters. ns, not significant. H , Aorta en face preparations from VE-cad +/+ and VE-cad GGG/GGG adult mice immunostained for VE-cad (red) and p120 (green). VE-cad levels at cell-cell junctions are significantly decreased in the VE-cad GGG/GGG p120-binding mutant, and p120 localization shifts from the cell-cell junctions to the cytoplasm. Scale bar: 20 μm. I , Quantitation of VE-cad and p120 levels at cell-cell junctions in the aortas of VE-cad +/+ and VE-cad GGG/GGG mice. Levels were quantitated from four independent experiments with 4-6 images per animal, and shown as the relative mean ± SEM. *p<0.05.

Article Snippet: Primary antibodies were: rat anti-VE-cad (BV13, eBioscience # 14144181, 1:500); rabbit anti-p120 (S-19, Santa Cruz Biotechnology, Inc #sc-1101, 1:250); mouse anti-beta-catenin (BD Biosciences #610153, 1:250); mouse anti-N-cadherin (BD Biosciences #610920, 1:250).

Techniques: Mutagenesis, Permeability, Staining, Quantitation Assay, Control, Binding Assay

Journal: bioRxiv

Article Title: VE-cadherin endocytosis controls vascular integrity and patterning during development

doi: 10.1101/769182

Figure Lengend Snippet: A , Normal levels of VE-cad at cell borders in VE-cad ΔJMD / ΔJMD endocytic mutant mice, despite lack of p120 binding. Aorta en face preparations from VE-cad +/+ and VE-cad ΔJMD / ΔJMD mice were immunostained for VE-cad (red) and p120 (green, top panels) or VE-cad (red) and β-catenin (green, bottom panels) and analyzed by confocal microscopy. Normal β-catenin at cell borders was also observed in VE-cad ΔJMD / ΔJMD mutants, corresponding to the normal VE-cad levels. Scale bar: 20 μm. B , Quantitation of protein levels at cell-cell junctions in the aortas of VE-cad +/+ and VE-cad ΔJMD / ΔJMD mice. No significant difference was detected in VE-cad or β-catenin levels between VE-cad +/+ and VE-cad ΔJMD / ΔJMD mice, whereas p120 was significantly decreased at cell junctions. Levels were quantitated from four independent experiments with 4-6 images per animal, and represent the relative mean ± SEM. *p<0.05 compared to VE-cad +/+ ; ns, not significant. C , VE-cad ΔJMD / ΔJMD mice from VE-cad ΔJMD /+ intercrosses were born at normal Mendelian ratios and displayed no defects in postnatal survival. The expected number of mice was calculated based on Mendelian genetics. p=0.24 in Χ 2 analysis. D , No increase in hemorrhages in E12.5 whole embryos (left) or P3 eye cups (right) in VE-cad ΔJMD / ΔJMD mutant mice compared to VE-cad +/+ controls. Scale bar (left): 1 mm Scale bar (right): 0.2 mm.

Article Snippet: Primary antibodies were: rat anti-VE-cad (BV13, eBioscience # 14144181, 1:500); rabbit anti-p120 (S-19, Santa Cruz Biotechnology, Inc #sc-1101, 1:250); mouse anti-beta-catenin (BD Biosciences #610153, 1:250); mouse anti-N-cadherin (BD Biosciences #610920, 1:250).

Techniques: Mutagenesis, Binding Assay, Confocal Microscopy, Quantitation Assay

Journal: bioRxiv

Article Title: VE-cadherin endocytosis controls vascular integrity and patterning during development

doi: 10.1101/769182

Figure Lengend Snippet: A , Immunostaining analysis for VE-cad (red) and p120 (green) on en face aorta preparations from VE-cad +/+ and VE-cad DEE/DEE adult mice. VE-cad levels at cell junctions appeared normal whereas p120 levels were partially decreased. Scale bar: 20 μm. B , Quantitation of VE-cad and p120 levels at cell-cell junctions in the aortas of VE-cad +/+ and VE-cad DEE/DEE mice. Levels were quantitated from five independent experiments with 4-6 images per animal, and shown as the relative mean ± SEM. *p<0.05. C , VE-cad DEE / DEE mice from VE-cad DEE /+ intercrosses were born at normal Mendelian ratios and displayed no defects in postnatal survival. p=0.49 in Χ 2 analysis. D , Whole embryos at E12.5 (left) and P3 eye cups (right) from VE-cad +/+ and VE-cad DEE/DEE mice. No intraretinal hemorrhaging was observed. Scale bar: 1 mm (left), 0.2 mm (right). E , No increase in vascular permeability in VE-cad DEE/DEE mutant mice in response to LPS treatment. Lung permeability was assessed in adult VE-cad +/+ and VE-cad DEE/DEE mice by the Evans blue dye method 6 hours after DPBS or LPS treatment. The bar graph represents means ± SEM with 7 mice per group. Two-way ANOVA **p<0.005; ns, not significant.

Article Snippet: Primary antibodies were: rat anti-VE-cad (BV13, eBioscience # 14144181, 1:500); rabbit anti-p120 (S-19, Santa Cruz Biotechnology, Inc #sc-1101, 1:250); mouse anti-beta-catenin (BD Biosciences #610153, 1:250); mouse anti-N-cadherin (BD Biosciences #610920, 1:250).

Techniques: Immunostaining, Quantitation Assay, Permeability, Mutagenesis

Journal: bioRxiv

Article Title: VE-cadherin endocytosis controls vascular integrity and patterning during development

doi: 10.1101/769182

Figure Lengend Snippet: A , Genotyping analysis of postnatal offspring from Tie-2-Cre + ; VE-cad DEE/+ ; p120 fl/fl × VE-cad DEE/+ ; p120 fl/fl matings. Less than expected numbers of Tie-2-Cre + ; VE-cad +/+ ; p120 fl/fl mice were born, based on expected Mendelian ratios, suggesting significant perinatal lethality in the presence of wild type VE-cad. However, Tie-2-Cre + ; VE-cad DEE/DEE ; p120 fl/fl mice were born near expected ratios, suggesting a rescue of lethality with disruption of the VE-cad endocytic motif. Genotyping was performed between P6-P8 and the expected number of mice was based on the total number of mice and Mendelian genetics. *p<0.05; ns, not significant in Χ 2 analysis. B , Quantitation of VE-cad levels at cell borders between adjacent p120 + or adjacent p120 − cells in the aortas of VE-cad-Cre + ; p120 fl/fl ; VE-cad +/+ (p120 CKO ; VE-cad +/+ ) and VE-cad-Cre + ; p120 fl/fl ; VE-cad DEE/DEE (p120 CKO ; VE-cad DEE/DEE ) mice as shown in panel C. VE-cad levels at p120 + cell borders were set to 100 and the percentage decrease in 120 − cells was quantitated. Graph represents the relative mean ± SEM, calculated from three mice per genotype **p<0.005 compared to p120 + ; ns, not significant. C , Rescue of VE-cad levels in p120-null cells by mutation of the DEE endocytic motif. Aorta en face immunostaining of p120 CKO ; VE-cad +/+ (top panels) or p120 CKO ; VE-cad DEE/DEE mice (bottom panels). Mosaic Cre-mediated deletion of p120 led to both p120 + (green) and p120 − cells within the same field of view. In p120 CKO ; VE-cad +/+ mice, VE-cad (red) levels were significantly decreased in p120 − cells, suggesting p120 is required for VE-cad membrane stability. In p120 CKO ; VE-cad DEE/DEE mice, no decreased in VE-cad levels were observed in p120 − cells, suggesting disruption of the DEE endocytic signal can stabilize VE-cad membrane levels in the absence of p120-binding. Scale bar: 25 μm.

Article Snippet: Primary antibodies were: rat anti-VE-cad (BV13, eBioscience # 14144181, 1:500); rabbit anti-p120 (S-19, Santa Cruz Biotechnology, Inc #sc-1101, 1:250); mouse anti-beta-catenin (BD Biosciences #610153, 1:250); mouse anti-N-cadherin (BD Biosciences #610920, 1:250).

Techniques: Disruption, Quantitation Assay, Mutagenesis, Immunostaining, Membrane, Binding Assay

Journal: bioRxiv

Article Title: Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission

doi: 10.1101/2025.09.15.676370

Figure Lengend Snippet: Fluorescein permeability assay of epithelial cell cultures in ALI at 0, 3, 9, and 12 days.

Article Snippet: Cells from the immortalized human epithelial cell line NuLi-1 (from ATCC) were expanded in a T25 flask for 4–5 days in serum-free Airway Epithelial Cell Basal Medium (BEGM; ATCC PCS-300-030) with additives from the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) as described .

Techniques: Permeability

Journal: bioRxiv

Article Title: Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission

doi: 10.1101/2025.09.15.676370

Figure Lengend Snippet: Confocal microscopy images of NuLi-1 lung epithelial cells in the ALI model after 12 days, stained for F-actin and with DAPI to highlight nuclei.

Article Snippet: Cells from the immortalized human epithelial cell line NuLi-1 (from ATCC) were expanded in a T25 flask for 4–5 days in serum-free Airway Epithelial Cell Basal Medium (BEGM; ATCC PCS-300-030) with additives from the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) as described .

Techniques: Confocal Microscopy, Staining