Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a UMAP plot displaying 14 identified cell types within the goat mammary glands. Cells are annotated and colored by type. Subsets of cell types including luminal, basal, fibroblast, immune, endothelial cell are labeled. b The percentages of −4W (red) and +1 W (blue) cells in each cell type are shown in a UMAP plot. c UMAP plots showing the expression of selected marker genes in four luminal subtypes. d Pseudotemporal trajectory analysis of scRNA-seq data of luminal secretory cells is shown in a UMAP plot. e Changes in the proportion of LumSecP and LumSec cells in luminal cell types were identified by scRNA-seq data at −4W and +1 W. n = 3 goats per group. f Representative images of tissue immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI at −4W and +1 W. Scale bars, 50 μm. g Bar plots exhibiting the percentage of ALDH1A3-positive cells in luminal cells (labeled by KRT18) in f . n = 15 sections per group. h Representative images of tissue immunofluorescence staining for FABP3 (red), KRT18 (green), and DAPI at −4W and +1 W. Scale bars, 50 μm. i Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in h . n = 20 sections per group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in e , g , i .

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Labeling, Expressing, Marker, Immunofluorescence, Staining

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Relative proportions of LumHR cells in total luminal cells identified by scRNA-seq data at −4W and +1 W. n = 3 goats per group. b , c Bar plots exhibiting the percentage of PGR - and ESR1 -positive cells within luminal cells in scRNA-seq data. n = 3 goats per group. d Representative images of immunofluorescence staining for PR (red), KRT18 (green), and DAPI (blue). Scale bars, 50 μm. e Bar plots exhibiting the percentage of PR-positive cells in luminal cells (labeled by KRT18) in ( d ). n = 5 goats per group. f Representative images of immunofluorescence staining for ER (red), KRT18 (green), and DAPI (blue). Scale bars, 50 μm. g Bar plots exhibiting the percentage of ER-positive cells in luminal cells (labeled by KRT18) in ( f ). n = 5 goats. h Violin plot showing the specific expression of PRLR in LumHR cells by scRNA-seq. i Heatmap displaying the transcriptional level of indicated genes related to milk protein and luminal differentiation in the goat mammary organoids ( n = 3 biological replicates) treated with or without prolactin and in the mammary tissue at −4W (non-lactation) and +1 W (lactation). n = 3 goats for tissues. j Proportions of luminal cell types in goat mammary organoids incubated with or without prolactin predicted by CIBERSORTx deconvolution. n = 3 biological replicates. k Representative images of immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI in mammary organoids incubated with or without prolactin. Scale bars, 10 μm. l Bar plots exhibiting the percentage of ALDH1A3-positive cells in luminal cells (labeled by KRT18) in ( k ). n = 14 domes in the control group and n = 10 domes in the prolactin treated group. m Representative images of immunofluorescence staining for FABP3 (red), KRT18 (green) and DAPI in mammary organoids incubated with or without prolactin. Scale bars, 10 μm. n Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in ( m ). n = 6 domes in the control group and n = 7 domes in the prolactin-treated group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in ( a – c , e , g , j , l , n ).

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Immunofluorescence, Staining, Labeling, Expressing, Incubation, Control

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Schematic illustration of targeted ablation of LumHR cells using the prlr -promoter to drive expression of DTA. b Experimental setup used in AAV intraductally injected mammary gland of ROSA-DTA +/ − mice under RR. c Whole-mount staining with carmine alum of mammary glands from ROSA-DTA +/ − mice (lactation day 2) intraductally injected with AAV-pPrlr-Cre or AAV-Control . Scale bars, 4 mm (top) and 500 μm (bottom). d , e Immunohistochemical staining and quantification of ER-positive luminal cells in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control. n = 3 mice per group. Scale bars, 50 μm. f , g Immunohistochemical staining and quantification of PR-positive luminal cells in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . n = 3 mice per group. Scale bars, 50 μm. h , i Immunohistochemical staining and quantification of β-casein-positive alveoli number per mm 2 in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . n = 3 mice per group. Scale bars, 50 μm. j Representative images of immunofluorescence staining for FABP3 (red), KRT18 (green), and DAPI in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . Scale bars, 50 μm. k Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in j . n = 3 mice per group. l Representative images of immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Contro l. Scale bars, 50 μm. m Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in ( l ). n = 3 mice per group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , g , i , l , m .

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Expressing, Injection, Staining, Control, Immunohistochemical staining, Immunofluorescence, Labeling

Journal: iScience

Article Title: JNJ-78306358, a first-in-class bispecific T cell engaging antibody targeting CD3 and HLA-G

doi: 10.1016/j.isci.2025.111876

Figure Lengend Snippet:

Article Snippet: Goat anti-hIgG1-AF647 (Fab’) 2 , Jackson ImmunoResearch , 109-606-098; RRID: AB_2337899.

Techniques: Recombinant, Lysis, Staining, Blocking Assay, Saline, Molecular Weight, Marker, Western Blot, Binding Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Polymer, Enzyme-linked Immunosorbent Assay, Activation Assay, Cell Culture, Membrane, Sterility

Journal: Scientific Reports

Article Title: ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

doi: 10.1038/srep28889

Figure Lengend Snippet: Immunohistochemistry analysis of paraffin fixed human subcutaneous fat tissue sections using specific antibodies against ( A ) ITGA5, ( B ) ITGAV and ( C ) fibronectin. Adipocytes can be morphologically identified as “signet ring cells” containing large fat vacuoles in the images. Protein expression was visualized using horseradish peroxidase (brown) and hematoxylin counterstaining (blue). Note the small ITGAV protein accumulations indicated by arrows at connecting adipocyte membranes ( B ). ( D ) Sorted ASCs and primary adipocytes of two representative donors (Donor#1 and Donor#2) were subjected to immunoblotting analyzing the expression of ITGA5, ITGAV and adipocyte specific marker FAPB4. ( E ) Cell lysates of in-vitro differentiated ASCs (day 14, left panel; proliferating cells are labeled with “prolif”, differentiated cells are labeled with “diff”) and the same cells separated according to their lipid droplet content (LD, right panel; lipid droplet containing cells are labeled with “+LD”, cells containing no lipid droplets are labeled with “−LD”) were subjected to immunoblotting detecting ITGAV, ITGA5, GAPDH and the adipocyte specific markers FABP4 and PLIN1. As loading controls of the depicted immunoblots cropped images of total protein stains acquired before blotting are shown. Full length gels are presented in .

Article Snippet: Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN ® TGX Stain-FreeTM Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21 Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany).

Techniques: Immunohistochemistry, Expressing, Western Blot, Marker, In Vitro, Labeling

Journal: Scientific Reports

Article Title: ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

doi: 10.1038/srep28889

Figure Lengend Snippet: ( A ) Representative microscopic images of knockdown and overexpressing cells subjected to in-vitro adipogenic differentiation for 14 days. Lipid droplets and nuclei were visualized by Oil Red O (ORO, red) and Hoechst 33342 (blue) staining of paraformaldehyde fixed cells. ( B ) The number of differentiated cells was determined by counting of differentiated cells per visual field. A minimum of 5 pictures was analyzed per group in each independent experiment. ( C ) Cells from the same experiment were stained with HCS-LipidTOX TM -Green and differentiation was analyzed by flow cytometry, determining extent of green fluorescence. Shown data represent the mean ± SD of 5 independent experiments. Data of KD-cells have been pooled. ( D ) Quantitative RT-PCR analysis of adipocyte marker genes ADIPOQ, FABP4/AP2, PLIN2 and PPARG was performed at day 14 of differentation, shown data represent the mean ± SEM of 3 independent experiments. Presented data of KD-cells have been pooled. Asterisks indicate p-values < 0.05.

Article Snippet: Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN ® TGX Stain-FreeTM Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21 Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany).

Techniques: Knockdown, In Vitro, Staining, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Marker

Journal: Scientific Reports

Article Title: ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

doi: 10.1038/srep28889

Figure Lengend Snippet: ( A ) Proliferation of cilengidite treated ASCs was assessed by Prestoblue ® proliferation assay 48 h after treatment. ( B ) Immunoblot analysis of ITGAV, ITGA5, ITGB3, survivin, p21 Cip1 , YAP and TAZ in ASCs treated with different concentrations of cilengitide for 48 h. ( C ) ASCs were subjected to adipogenic differentiation for 14 days and different concentrations of cilengitide were added to the medium. Extent of differentiation was visualized by Oil Red O staining (ORO) of paraformaldehyde fixed cells and assessed by counting differentiated cells per visual field. Asterisks indicate p-values < 0.05, a minimum of 5 pictures per group were analyzed in each experiment. Shown data represent the mean ± SD of 3 independent experiments. ( D ) Representative microscopic images of cilengitide or DMSO treated ASCs subjected to in-vitro differentiation for 14 days showing ORO stained lipid droplets (red) and Hoechst stained nuclei (blue), scale bar: 50 μm. ( E ) Cell lysates of day 14 differentiated ASC exposed to 10 μM cilengitide or control treatment (DMSO) were subjected to immunoblotting for expression analysis of the adipocyte specific markers FABP4 and PLIN1. As loading controls of the depicted immunoblots cropped images of total protein stains acquired before blotting are shown.

Article Snippet: Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN ® TGX Stain-FreeTM Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21 Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany).

Techniques: Proliferation Assay, Western Blot, Staining, In Vitro, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Development of Sjogren's syndrome in nonobese diabetic-derived autoimmune-prone C57BL/6.NOD-Aec1Aec2 mice is dependent on complement component-3.

doi: 10.4049/jimmunol.179.4.2318

Figure Lengend Snippet: FIGURE 6. Detection of anti-M3R Abs. Pooled sera collected from C57BL/6 (27 wk of age), C57BL/6.NOD-Aec1Aec2 (30 wk of age), and C57BL/ 6.NOD-Aec1Aec2.C3/ (24 wk of age) mice. Mouse M3R-transfected Flp-In CHO cells were incubated with either a pooled sera or respective isotype control sera. Cells were washed with FACS buffer, resuspended in 50 l of FACS buffer and developed with either FITC-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG2c, or IgG3, then analyzed using a FACScan cytometer. M (marker) values equal mean fluorescent indices.

Article Snippet: Cells were washed once with FACS buffer, resuspended in 50 l of FACS buffer, and incubated for 30 min at 4°C with either FITCconjugated goat anti-mouse IgG1, IgG2b, IgG2c, IgG3, IgM, IgA, IgE, and IgD (Southern Biotechnology Associates).

Techniques: Transfection, Incubation, Control, Cytometry, Marker