Additional file 2: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Tissue digestion and cell fraction separation protocol. (PDF 231 kb

Additional file 4: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Figure S1. Immunofluorescence of glucocorticoid receptor in myoepithelial, MCF10A and MCF10DCIS cells. Hoechst was used to counterstain nuclei. Scale bar= 20 µm. (PPTX 917 kb

Additional file 7: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Figure S4. Representative immunofluorescence images of MCF10DCIS xenografts in chicken embryo CAM membrane.a Hoechst in blue, p63 in red and laminin in green. b Merge of p63 and laminin double immunofluorescence images and zoom in showing an acini detail. Scale bar=20 µm. (PPT 4617 kb

Additional file 6: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Figure S3. Corticosterone effects on mammary epithelial cells viability, apoptosis and functional abilities. a MCF10A epithelial, primary myoepithelial cells and MCF10DCIS cell viability evaluated by MTT assay after 48h of increasing doses of corticosterone treatment (0.125-1.5 µM). b MCF10A epithelial, primary myoepithelial cells and MCF10DCIS determination of cell apoptosis in its different stages (early, apoptosis, late and total) after treatment with corticosterone 0-1 µM by the Annexin V method and measured by flow cytometry. All experiments were carried out in triplicate. Statistical analysis was made using ANOVA followed by Dunn’s multiple test. c and d. Influence of corticosterone treatment on 3D growth of primary epithelial and myoepithelial cells and on MCF10DCIS for 14 days by immunofluorescence. Treatment with corticosterone 1 µM or vehicle (methanol) was carried out from day 5 after seeding until day 14. c Upper part. Immunodetection of K14 (myoepithelial cells), K19 (epithelial cells), with hoechst used as nuclei counterstaining. Scale bar=50 µm. c Bottom part. Quantification of morphometric analysis in control group and corticosterone-treated group of number of acini formed and related quantification of disrupted acini per total number of acini. d Upper part. Immunofluorescence of laminin (basement membrane), K14 (myoepithelial cells) and hoechst to counterstain nuclei. Scale bar=100 µm. d Bottom part. Quantification of laminin intensity after treatment with corticosterone 1 µM or vehicle by Image J software comparison test. e Immunofluorescence in MCF10DCIS 3D growth of laminin (basement membrane), Muc1 (epithelial cells) and hoechst to stain the nuclei. Arrows indicated rupture points of the acini showed. F. Morphometric quantification of disrupted acini and acinar fusion and intensity of laminin determined by integrated density parameter of Image J software. Scale bar=50µm. All experiments were carried out in triplicate. Statistical analysis was made using the Mann-Whitney test. (PPTX 3174 kb

Additional file 5: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Figure S2. In vivo progression of MCF10DCIS xenografts. a Histology of tumours (H&E) and expression of α-SMA and p63 were analysed at the indicated time points after injection (n=5 animals). Scale bar= 100 µm. b Representative image of glucocorticoid receptor immunohistochemistry in in vivo samples. Scale bar= 20 µm. Red arrow indicates myoepithelial positive cells for GR and black arrow shows positivity in epithelial cells. (PPTX 5642 kb

Additional file 3: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Table S2. Description of patient samples used to confirm apoptosis in human myoepithelial cell samples. Internal codes, histopathological description and receptor characteristics of patient samples. ND: not detectable. NA: not applicable. A: amplified. (PPTX 90 kb

Additional file 1: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Table S1. Description of patient samples used for the primary mammary epithelial and myoepithelial cells. Internal codes, histological description and age of patients. (PPTX 53 kb