Journal: Breast Cancer Research : BCR

Article Title: Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

doi: 10.1186/s13058-024-01851-4

Figure Lengend Snippet: Effect of Itga6 deletion on growth and differentiation of Brca1/p53-deficient tumors. A Immunofluorescent staining with anti-Itgα6 (white), and anti-Ki67 (red) antibodies. The graph shows the percentage of Ki67 + cells (mean ± SEM) in small (< 750 mm 3 ; n = 8 animals per genotype) and large (> 750 mm 3 ; n = 7 animals per genotype) tumors. B Immunofluorescent staining with anti-K8 (white), and anti-cleaved caspase 3 (CC3, red) antibodies. The graph shows the percentage of CC3 + cells (mean ± SEM) in small (< 750 mm 3 ; n = 4 animals per genotype) and large (≥ 750 mm 3 ; n = 4 animals per genotype) tumors. A and B : Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. C Tumor growth curves of Brca1p53-KO (n = 7) and α6KO/Brca1p53-KO (n = 7) tumors. The difference between groups is not significant (Kruskal–Wallis test). D Upper panels: Representative FACS analysis of a Brca1p53-KO and a α6KO/Brca1p53-KO tumor with CD24 and Lin (CD31/CD45/Ter-119) antibodies. The red rectangle shows the CD24 + Lin − population sorted for gene expression analysis. Lower panels: Representative FACS analysis of tumors with CD24 and CD49f (α6-integrin) antibodies showing depletion of surface Itgα6 in most epithelial cells of the α6KO/Brca1p53-KO tumor. E RT-qPCR analysis of the CD24 + Lin − tumor cell population from Brca1p53-KO (n = 5) and α6KO/Brca1p53-KO (n = 5) animals (mean ± SEM). F Representative immunofluorescent staining with anti-K5 (red) and anti-K8 (white) antibodies. Magnification of the white rectangle is shown in the right panels of Brca1p53-KO and α6KO/Brca1p53-KO tumors. Nuclear DAPI staining is shown in blue. Scale bar: 75 µm (left panels for each tumor) and 20 µm (magnification in the right panels). In A , B , E * P < 0.05, n.s., non-significant

Article Snippet: The primers used for RT-qPCR analysis were purchased from SABiosciences/Qiagen or designed using Oligo 6.8 software (Molecular biology Insights) and synthesized by Eurogentec.

Techniques: Staining, Expressing, Quantitative RT-PCR

Journal: Breast Cancer Research : BCR

Article Title: Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

doi: 10.1186/s13058-024-01851-4

Figure Lengend Snippet: The aberrant expression of basal/EMT-like genes in luminal progenitors of Brca1/p53-deficient mice is reduced by Itga6 deletion. A Hematoxylin and Eosin staining of juxta-tumoral mammary gland sections from Brca1p53-KO and α6KO/Brca1p53-KO mice (bearing a tumor in another gland) and a control littermate. Scale bar: 100 µm. B Immunofluorescent staining with anti-K8 (white), and anti-Ki67 (red) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of Ki67 + cells (mean ± SEM) obtained from 4 controls, 6 Brca1p53-KO and 6 α6KO/Brca1p53-KO mice. C , D Representative FACS analysis of juxta-tumoral glands from Brca1p53-KO and α6KO/Brca1p53-KO mice. C separation of basal (green) and luminal (orange) populations. Right: graph showing the percentage of basal and luminal cells in the Lin- population in 5 independent cell sorting experiments. D Analysis of ICAM1 expression gated in the luminal cells. Right: graph showing the percentage of luminal ICAM1 + and ICAM1- cells relative to the total luminal population in five independent cell sorting experiments. E RT-qPCR analysis of the ICAM1 + LP from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands and control glands (5 animals per group) The graphs present mean ± SEM. F Immunofluorescent staining with anti-K8 (green), and anti-K14 (red) antibodies of control normal gland and normal-looking ducts (upper panels), or of mammary hyperplasic lesions (lower panels) developed in Brca1p53-KO and α6KO/Brca1p53-KO mice. In normal ducts, dashed rectangles indicate magnifications shown in the right panels. Note the presence of numerous cells co-expressing K8 and K14, indicated by white arrowheads in normal ducts, or marked in yellow in lesions (lower panels). Nuclear DAPI staining is shown in blue. Scale bar: 20 µm (12 µm in magnifications). G Western blot analysis of ICAM1 + LP isolated from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands. Control cells are shown for comparison. In B – E , * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., non-significant

Article Snippet: The primers used for RT-qPCR analysis were purchased from SABiosciences/Qiagen or designed using Oligo 6.8 software (Molecular biology Insights) and synthesized by Eurogentec.

Techniques: Expressing, Staining, FACS, Quantitative RT-PCR, Western Blot, Isolation, Comparison

Journal: Breast Cancer Research : BCR

Article Title: Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

doi: 10.1186/s13058-024-01851-4

Figure Lengend Snippet: Induction of the cell cycle inhibitor p16 in the Brca1/p53-deficient preneoplastic glands. A Immunofluorescent staining with anti-p16 (red), anti-K8 (white) and anti α-SMA (green) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. B RT-qPCR analysis of Cdkn2a expression (coding for the p16 protein) in ICAM1 + LP cells (n = 4) and CD24 + tumor cells (n = 3) from Brca1p53-KO and α6KO/Brca1p53-KO mice at different stages. Control ICAM1 + LP values are shown for comparison (n = 4). C Immunofluorescent staining with anti-p16 (red) and anti-K8 (white) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of p16 + cells (mean ± SEM) obtained from 3–4 animals per group. D Western blot analysis of luminal progenitor cells isolated from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands. Cells isolated from control littermate females are shown for comparison. E Immunofluorescent staining with anti-p16 (red), and anti-Ki67 (green) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the fraction of p16 + that are also Ki67 + (mean ± SEM) obtained from 3–4 animals per group. F Immunofluorescent staining with anti-phospho-Rb (red), and anti-K8 (white) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of phospho-Rb + luminal (K8 +) cells (mean ± SEM) obtained from 4 animals per group. In B , C , E , F , * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., non-significant

Article Snippet: The primers used for RT-qPCR analysis were purchased from SABiosciences/Qiagen or designed using Oligo 6.8 software (Molecular biology Insights) and synthesized by Eurogentec.

Techniques: Staining, Quantitative RT-PCR, Expressing, Comparison, Western Blot, Isolation