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 Violin plot displaying the expression level of top transcription factors (TFs) in luminal subtypes at −4W and +1W. b Heatmap showing the regulon activities of the top TFs in luminal subtypes at −4W. c Immunohistochemical staining for IRF1 in the goat mammary gland at −4W and +1W. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. d Quantification of IRF1-positive cells in c . n = 8 sections from 4 goats. e Representative images of immunohistochemical staining for PR in the goat mammary organoids treated with or without IFNγ. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. f Quantification of PR-positive cells in e . n = 5 domes per group. g Representative images of carmine-stained mammary gland whole mounts in WT and IRF1-KO mice at 9 weeks. Scale bars, 0.4 mm. h − k Automatic quantification of the number junctions ( h ), tips ( i ), branches ( j ) and lumen diameters ( k ) of mammary tissues in f . n = 6 mice in wild type and n = 3 in IRF1-KO mice. n = 30 and n = 15 ductal lumens in WT and IRF1-KO mice, respectively. l , m Immunohistochemical staining ( l ) and quantification ( m ) of PR and ER in mammary tissues from WT or IRF1-KO mice at 9 weeks. Nuclei were counterstained with hematoxylin ( l ). n = 4 mice per group. Scale bars, 10 μm. n , o Immunohistochemical staining ( n ) and quantification ( o ) of PR and ER in mammary tissues from WT or IRF1-KO mice during RR. Nuclei were counterstained with hematoxylin. n = 5 mice per group. Scale bars, 10 μm. p Pre-ranked GSEA graphical output for the enrichment in IRF1-KO mice mammary glands of the gene set estrogen response early from the Molecular Signatures Database Hallmarks collection. n = 3 mice per group. q Heatmap representing the log 2 fold change expression of hormone-driven genes in IRF-KO compared to WT at 9 weeks. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , f , h – k , m , o .

Article Snippet: For immunohistochemistry, goat mammary tissues were stained with IRF1 (homemade anti-goat rabbit polyclonal antibody) and ESRRB (#22644-1-AP, ProteinTech).

Techniques: Expressing, Immunohistochemical staining, 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 The genomic loci with IRF1 motifs are selected and the ATAC-seq signal intensity is shown in heatmaps. The average signal intensity is shown on top. b Heatmap displaying the transcriptional level of genes presumably bound by IRF1. n = 3 goats per group. c Heatmaps showing the signal intensity of IRF1 CUT&Tag in goat mammary tissues at −4W and +1 W. The average signal intensity is shown on top. d ATAC-seq and IRF1 CUT&Tag profiles at the ESRRB locus in −4W and +1 W are shown. The differential regions between −4W and +1 W with IRF1 motifs are highlighted in yellow. e UMAP plot showing the specific expression of ESRRB in LumHR cells by scRNA-seq data. f , g Immunohistochemical staining and quantification of ESRRB in goat mammary tissues at −4W and +1 W. Representative images of Immunohistochemical staining ( f ). Nuclei are counterstained with hematoxylin. n = 10 sections from 5 goats per group. Scale bars, 20 μm. Two-sided Student’s t -test. h Luciferase reporter assays in goat mammary epithelial cells. Cells are transfected with WT IRF1 motif (IRF1-MWT) or IRF1-motif site mutation (IRF1-MM) vector and treated with IFNγ or not. n = 4 biological replicates. Two-way ANOVA test. i , j Immunohistochemical staining and quantification of ESRRB in mouse WT or IRF1-KO mammary tissues under RR. Representative images of Immunohistochemical staining ( i ). Nuclei are counterstained with hematoxylin. n = 4 mice per group. Scale bars, 50 μm. Two-sided Student’s t -test. k The proposed model in the current study is that a reduction of LumHR cells triggered by IRF1-ESRRB signaling upregulation promotes the accumulation of LumSecP during RR in ruminants. LumHR cells control the differentiation of LumSecP to LumSec cells through the PRLR pathway and regulate the cell composition of luminal lineages during RR. Created with BioRender.com.

Article Snippet: For immunohistochemistry, goat mammary tissues were stained with IRF1 (homemade anti-goat rabbit polyclonal antibody) and ESRRB (#22644-1-AP, ProteinTech).

Techniques: Expressing, Immunohistochemical staining, Staining, Luciferase, Transfection, Mutagenesis, Plasmid Preparation, 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 Violin plot displaying the expression level of top transcription factors (TFs) in luminal subtypes at −4W and +1W. b Heatmap showing the regulon activities of the top TFs in luminal subtypes at −4W. c Immunohistochemical staining for IRF1 in the goat mammary gland at −4W and +1W. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. d Quantification of IRF1-positive cells in c . n = 8 sections from 4 goats. e Representative images of immunohistochemical staining for PR in the goat mammary organoids treated with or without IFNγ. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. f Quantification of PR-positive cells in e . n = 5 domes per group. g Representative images of carmine-stained mammary gland whole mounts in WT and IRF1-KO mice at 9 weeks. Scale bars, 0.4 mm. h − k Automatic quantification of the number junctions ( h ), tips ( i ), branches ( j ) and lumen diameters ( k ) of mammary tissues in f . n = 6 mice in wild type and n = 3 in IRF1-KO mice. n = 30 and n = 15 ductal lumens in WT and IRF1-KO mice, respectively. l , m Immunohistochemical staining ( l ) and quantification ( m ) of PR and ER in mammary tissues from WT or IRF1-KO mice at 9 weeks. Nuclei were counterstained with hematoxylin ( l ). n = 4 mice per group. Scale bars, 10 μm. n , o Immunohistochemical staining ( n ) and quantification ( o ) of PR and ER in mammary tissues from WT or IRF1-KO mice during RR. Nuclei were counterstained with hematoxylin. n = 5 mice per group. Scale bars, 10 μm. p Pre-ranked GSEA graphical output for the enrichment in IRF1-KO mice mammary glands of the gene set estrogen response early from the Molecular Signatures Database Hallmarks collection. n = 3 mice per group. q Heatmap representing the log 2 fold change expression of hormone-driven genes in IRF-KO compared to WT at 9 weeks. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , f , h – k , m , o .

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, 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 The genomic loci with IRF1 motifs are selected and the ATAC-seq signal intensity is shown in heatmaps. The average signal intensity is shown on top. b Heatmap displaying the transcriptional level of genes presumably bound by IRF1. n = 3 goats per group. c Heatmaps showing the signal intensity of IRF1 CUT&Tag in goat mammary tissues at −4W and +1 W. The average signal intensity is shown on top. d ATAC-seq and IRF1 CUT&Tag profiles at the ESRRB locus in −4W and +1 W are shown. The differential regions between −4W and +1 W with IRF1 motifs are highlighted in yellow. e UMAP plot showing the specific expression of ESRRB in LumHR cells by scRNA-seq data. f , g Immunohistochemical staining and quantification of ESRRB in goat mammary tissues at −4W and +1 W. Representative images of Immunohistochemical staining ( f ). Nuclei are counterstained with hematoxylin. n = 10 sections from 5 goats per group. Scale bars, 20 μm. Two-sided Student’s t -test. h Luciferase reporter assays in goat mammary epithelial cells. Cells are transfected with WT IRF1 motif (IRF1-MWT) or IRF1-motif site mutation (IRF1-MM) vector and treated with IFNγ or not. n = 4 biological replicates. Two-way ANOVA test. i , j Immunohistochemical staining and quantification of ESRRB in mouse WT or IRF1-KO mammary tissues under RR. Representative images of Immunohistochemical staining ( i ). Nuclei are counterstained with hematoxylin. n = 4 mice per group. Scale bars, 50 μm. Two-sided Student’s t -test. k The proposed model in the current study is that a reduction of LumHR cells triggered by IRF1-ESRRB signaling upregulation promotes the accumulation of LumSecP during RR in ruminants. LumHR cells control the differentiation of LumSecP to LumSec cells through the PRLR pathway and regulate the cell composition of luminal lineages during RR. Created with BioRender.com.

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining, Luciferase, Transfection, Mutagenesis, Plasmid Preparation, Control