Journal: PLoS ONE

Article Title: Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells

doi: 10.1371/journal.pone.0272819

Figure Lengend Snippet: HC11 cells were seeded in base media for 24 hours (UD). HC11 cells were seeded and supplemented with EGF and INS for 3 days (UD+EGF). Finally, HC11 cells were differentiated in base media containing PRL, INS and DEX, and allowed to proceed for 4 days in the absence (DF) or presence of the vehicle (MeOH). Cells from all conditions were processed for total RNA or protein isolation, as described in methods. The relative (compared to RPL0 and RPL8 ) mRNA levels of CSN2 (A) and WAP (B) were determined. Gene expression analysis was repeated with 6 distinct replicates. Protein expression of β-casein was assessed, as described in methods, using 15μg of total protein for each sample. (C) A representative blot of β-casein expression is shown along with a bar graph quantifying its expression, normalized to β-actin, for undifferentiated (UD), EGF supplemented (UD+EGF), differentiated (DF) and differentiated in the presence of vehicle (MeOH), based on 4 replicates. Lipid levels were assessed in undifferentiated cells (UD) and differentiated cells (DF), by conducting an Oil Red O (ORO) assay, as described in methods. Representative images of ORO-stained undifferentiated HC11 cells (UD) and differentiated cells (DF), taken with a phase-contrast microscope (20x magnification). (F) Lipid levels were quantified in cells, as described in the methods, from 6 distinct replicates of undifferentiated HC11 cells (UD) and differentiated cells (DF). Results were plotted as mean ± SEM and compared using either a student’s t-test (for groups ≤2), or one-way ANOVA (for groups ≥ 3). Statistically significant changes were represented by distinct letters on bar graphs.

Article Snippet: Anti-β-casein mAb , Santa Cruz Biotechnology , sc-166684 , 1:1000.

Techniques: Isolation, Gene Expression, Expressing, Oro Assay, Staining, Microscopy

Journal: PLoS ONE

Article Title: Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells

doi: 10.1371/journal.pone.0272819

Figure Lengend Snippet: HC11 cells were seeded in base media for 24 hours (UD) and processed for total RNA or protein, as described in the methods. HC11 cells were differentiated in base media containing PRL, INS and DEX, as described in methods, and treated with vehicle control (MeOH), 10μM THC or 10μM CBD for 4 days, and processed for total RNA or protein. The relative mRNA levels of CSN2 (A) and WAP (B) were determined. Gene expression analysis was repeated with 6 distinct replicates. Milk protein expression was assessed, as described in the methods, using 15μg of total protein for each sample. Representative blots of β-casein (C) and WAP (B) are shown along with a bar graph quantifying the protein expression, normalized to β-actin, for undifferentiated cells (UD), vehicle control (MeOH), 10μM THC and 10μM CBD treated HC11 cells, based on 4 replicates. Results were plotted as mean ± SEM and compared using one-way ANOVA. Statistically significant changes were represented by distinct letters.

Article Snippet: Anti-β-casein mAb , Santa Cruz Biotechnology , sc-166684 , 1:1000.

Techniques: Control, Gene Expression, Expressing

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

Journal: Scientific Reports

Article Title: Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

doi: 10.1038/srep43020

Figure Lengend Snippet: Protein-protein interaction network of the identified proteins from co-immunoprecipitation with antibody β-casein ( a ), casein micelle in Holstein ( b ) generated with STRING software. Each node represents a protein; different line colors represent the types of evidence for the association: pink lines from experimental study, the blue lines from databases, and the yellow lines from abstracts of articles published in PubMed.

Article Snippet: Monoclonal antibodies against β-casein of cows were prepared by the Beijing Protein Institute (Beijing, China).

Techniques: Immunoprecipitation, Generated, Software

Journal: Scientific Reports

Article Title: Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

doi: 10.1038/srep43020

Figure Lengend Snippet: Western blot analysis of β-casein ( a ) and ‘far-western blot’ analysis of β-casein interacted with major whey proteins ( b ). M shows protein marker; Lanes 1, 2, and 3 represent samples with 9, 18 and 36 μg milk proteins; Lanes 4 shows β-casein standard.

Article Snippet: Monoclonal antibodies against β-casein of cows were prepared by the Beijing Protein Institute (Beijing, China).

Techniques: Western Blot, Far Western Blot, Marker