anti-mouse β-casein  (Covalab Inc)

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Characterization of Organoid Cultures to Study the Effects of Pregnancy Hormones on the Epigenome and Transcriptional Output of Mammary Epithelial Cells

doi: 10.1007/s10911-020-09465-0

Figure Lengend Snippet: Pregnancy hormones induce robust upregulation of Casein 2 (Csn2) in post-pregnancy mammary organoid cultures. ( a ) Genome browser tracks showing distribution of H3K27Ac peaks at the Csn2 locus in luminal ductal MECs harvested from pre- and post-pregnancy mice at distinct pregnancy cycles. ( b ) mRNA levels (qPCR) of casein genes, Csn2 and Csn3 , in pre- and post-pregnancy mammary organoids grown with essential medium, complete medium (supplemented with pregnancy hormones) or complete medium with FGF2 n = 2 biological replicates, with 3 technical replicates each. *** p = 0.0002, ** p = 0.002, * p = 0.01 ( c ) Csn2 mRNA levels (2^deltadeltaCT) in pre-pregnancy mammary organoids grown with complete medium. Normalized to pre- pregnancy organoids treated with complete medium for 3 h. ( d ) Immunofluorescence (IF) imaging of whole mounted pre- and post-pregnancy mammary organoids before or after complete medium exposure for 12 h and 72 h. KRT8 (blue), KRT5 (red) and CSN2 (green). Scale: 100 μm. For analyses, error bars indicate standard error of the mean (SEM) across samples of same experimental group. p values were defined using Welch’s t-test

Article Snippet: Antibodies for IF: anti-β-casein/csn2 mouse monoclonal antibody (Santa Cruz Biotechnology Inc., #sc-166,530, 200 μg/mL, 1:300 dilution, RRID: AB_2084348), anti-Cytokeratin5 rabbit monoclonal antibody (Abcam, #EP1601Y, 0.5 mg/ML 1:300 dilution RRID:AB_869890), anti-Cytokeratin-8 mouse monoclonal antibody (Abcam, #EP1628Y, 0.5 mg/mL 1:300 dilution, RRID:AB_869901).

Techniques: Immunofluorescence, Imaging

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Characterization of Organoid Cultures to Study the Effects of Pregnancy Hormones on the Epigenome and Transcriptional Output of Mammary Epithelial Cells

doi: 10.1007/s10911-020-09465-0

Figure Lengend Snippet: Utilization of organoid cultures to define players in pregnancy-induced development and post-pregnancy epigenome. ( a ) Representative brightfield images of mammary organoids treated with essential medium or complete medium, supplemented with either DMSO (Dimethyl sulfoxide, control), or EZH2 inhibitor UNC1999 for 48 h. Arrows indicated examples of branching organoids. Scale = 200 μm. ( b ) Branching quantification of pre- and post-pregnancy mammary organoid cultures treated with essential medium or complete medium, supplemented with either DMSO (control) or EZH2 inhibitor UNC1999. 13 fields of view per well/replicate. n = 25 organoids. ns = not significant; * p = 0.027 differences between post-pregnancy organoids treated with complete media and complete media with UNC1999. Error bars indicate standard error of the mean (SEM) across samples of same experimental group. p values were defined using Students t-test. ( c ) Size quantification of pre- and post-pregnancy mammary organoid cultures treated with essential medium or complete medium, supplemented with either DMSO (control) or EZH2 inhibitor UNC1999. n = 20 organoids per condition. ns = not significant; * p = 0.018 differences between post-pregnancy organoids treated with complete medium and DMSO and post-pregnancy organoids treated with complete medium and UNC1999. For analyses, error bars indicate standard error of the mean (SEM) across samples of same experimental group. p values were defined using Welch’s t-test. ( d ) Csn2 mRNA levels (qPCR) in pre- and post-pregnancy mammary organoid cultures treated with either essential medium or complete medium, with DMSO control or UNC1999. * p = 0.0364 differences between post-pregnancy organoids treated with complete medium and DMSO and post pregnancy organoids treated with complete medium and UNC1999. *** p = 0.0009 differences between pre-pregnancy organoids treated with complete medium and UNC1999, and post-pregnancy organoids treated with complete medium and UNC1999. Error bars indicate standard error of the mean (SEM) across samples of same experimental group. p values were defined using Welch’s t-test

Article Snippet: Antibodies for IF: anti-β-casein/csn2 mouse monoclonal antibody (Santa Cruz Biotechnology Inc., #sc-166,530, 200 μg/mL, 1:300 dilution, RRID: AB_2084348), anti-Cytokeratin5 rabbit monoclonal antibody (Abcam, #EP1601Y, 0.5 mg/ML 1:300 dilution RRID:AB_869890), anti-Cytokeratin-8 mouse monoclonal antibody (Abcam, #EP1628Y, 0.5 mg/mL 1:300 dilution, RRID:AB_869901).

Techniques: Control