human mammary epithelial cell line mcf10a  (ATCC)

Journal: Epigenetics

Article Title: SIRT1/DNMT3B-mediated epigenetic gene silencing in response to phytoestrogens in mammary epithelial cells.

doi: 10.1080/15592294.2025.2473770

Figure Lengend Snippet: Figure 1. Impact of pterostilbene (PTS) or genistein (GEN) on cell growth and SIRT1 expression in MCF10A mammary epithelial cells. (a,b) MCF10A cell growth in response to different PTS (a) or GEN (b) concentrations upon 4- and 9-day exposure. Bar charts show MCF10A cell growth in response to 9-day exposure to 7 μM and 11 μM PTS (a) or 8 μM and 22 μM GEN (b), as compared with vehicle- treated cells (ctrl, ethanol for PTS and DMSO for GEN). All results were determined by the trypan blue exclusion test, and cell number

Article Snippet: Cell culture and treatment with pterostilbene (PTS) and genistein (GEN) The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA).

Techniques: Expressing

Journal: Epigenetics

Article Title: SIRT1/DNMT3B-mediated epigenetic gene silencing in response to phytoestrogens in mammary epithelial cells.

doi: 10.1080/15592294.2025.2473770

Figure Lengend Snippet: Figure 2. SIRT1 binding at RNF114, RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells. (a-c) SIRT1 binding after 9-day exposure to 7 μM and 11 μM PTS at RNF114 (a), RNF169 (b), and rDNA (c) promoters. (d-f) SIRT1 binding after 9-day exposure to 8 μM and 22 μM GEN at RNF114 (d), RNF169 (e), and rDNA (f) promoters. All results were determined by quantitative chromatin immunoprecipitation (qChIP), and represent mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: Cell culture and treatment with pterostilbene (PTS) and genistein (GEN) The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA).

Techniques: Binding Assay, Chromatin Immunoprecipitation

Journal: Epigenetics

Article Title: SIRT1/DNMT3B-mediated epigenetic gene silencing in response to phytoestrogens in mammary epithelial cells.

doi: 10.1080/15592294.2025.2473770

Figure Lengend Snippet: Figure 3. PTS- and gen-mediated changes in gene expression and DNA methylation of target genes in MCF10A cells. (a-b) expression at mRNA levels for RNF114, RNF169 and rDNA upon 9-day exposure to 7 μM and 11 μM PTS (a), and 8 μM and 22 μM GEN (b) in MCF10A cells as determined by qRT-pcr. (c Maps of promoter regions of RNF169 and rDNA, where the light blue shaded area represents the CpG sites tested by pyrosequencing (numbered CpGs). (d-e) average methylation status of CpG sites within RNF169 and rDNA promoters in MCF10A cells after 9-day exposure to 7 μM and 11 μM PTS (d) or 8 μM and 22 μM GEN (e), as determined by pyrosequencing. All results represent mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: Cell culture and treatment with pterostilbene (PTS) and genistein (GEN) The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA).

Techniques: Gene Expression, DNA Methylation Assay, Expressing, Quantitative RT-PCR, Methylation

Journal: Epigenetics

Article Title: SIRT1/DNMT3B-mediated epigenetic gene silencing in response to phytoestrogens in mammary epithelial cells.

doi: 10.1080/15592294.2025.2473770

Figure Lengend Snippet: Figure 4. DNMT3B binding and SIRT1-DNMT3B co-occupancy at RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells. (a,b) DNMT3B binding after 9-day exposure to 7 μM and 11 μM PTS at RNF169 (a) and rDNA (b) promoters. (c,d) DNMT3B binding after 9-day exposure to 8 μM and 22 μM GEN at RNF169 (c) and rDNA (d) promoters. All results were determined by quantitative chromatin immunoprecipitation (qChIP). (e) ChIP-re-ChIP assay was used to determine co-occupancy of SIRT1 and DNMT3B at RNF169 and rDNA promoters in response to 9-day exposure to 22 μM GEN. ‘Double: SIRT1-DNMT3B’ refers to SIRT1 binding first followed by DNMT3B binding; ‘double: DNMT3B-SIRT1’ indicates the reverse order. The sequential binding orders at the same region can be identified. Results were quantified by quantitative PCR. All results represent mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: Cell culture and treatment with pterostilbene (PTS) and genistein (GEN) The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Epigenetics

Article Title: SIRT1/DNMT3B-mediated epigenetic gene silencing in response to phytoestrogens in mammary epithelial cells.

doi: 10.1080/15592294.2025.2473770

Figure Lengend Snippet: Figure 5. Expression of SIRT1, RNF169 and rDNA upon 9-day exposure to 22 μM GEN in MCF10A cells upon SIRT1 depletion. MCF10A cells were transfected with control siRNA and siRNA targeting SIRT1 followed by exposure to 22 μM GEN. (a-b) expression of SIRT1 at mRNA (a) and protein (b) level, as determined by qRT-pcr and western blot, respectively. (c-d) expression of RNF169 (c), and rDNA (d) at mRNA levels, as determined by qRT-pcr. All results represent mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: Cell culture and treatment with pterostilbene (PTS) and genistein (GEN) The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity

doi: 10.1093/nar/gkae1143

Figure Lengend Snippet: Cell type and context-dependent effect on p63-dependent CRE activity. ( A ) Western blot analysis of p63 expression in MCF10A TP53 −/− , HaCaT and SCC25 cells. GAPDH is used as a loading control. ( B ) Distribution of p63-dependent CRE function in MCF10A TP53 −/− , HaCat or SCC25 cells ( n = 9 697). ( C ) Sankey diagram derived from ( B ) depicting changes in p63-dependent CRE function across three cell types. Numbers indicate CRE counts in each group and cell line. ( D–E ) Dot plots highlighting TF motifs enriched in p63-activated CREs that are shared ( D ) between SCC25 and MCF10A TP53 −/− cell lines or uniquely enriched in each ( E ). Dot size represents fold-change enrichment over background. Color scale indicates log-transformed inverted P -value. ( F ) Gene expression analysis of selected TFs from MCF10A TP53 −/− and SCC25 cells ( , ). Differential gene expression between the two cell types is represented by the fold-change and adjusted P-value as determined by DESeq2, and indicated in the legend.

Article Snippet: All human mammary epithelial cell lines MCF10A TP53+/+ and TP53 −/− (Sigma-Millipore clls1049) were cultured in 1:1 Dulbecco's Modified Eagle's Medium: Ham's F-12 (Gibco, #11330–032), supplemented with 5% Horse Serum, (Gibco, #16050–122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100–15), 0.5 μg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 μg/mL insulin (Sigma, #I-1882) and 1% penicillin-streptomycin (Gibco, #15240–062).

Techniques: Activity Assay, Western Blot, Expressing, Control, Derivative Assay, Transformation Assay, Gene Expression

Journal: Nucleic Acids Research

Article Title: Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity

doi: 10.1093/nar/gkae1143

Figure Lengend Snippet: Relationship between meta-analysis-based p63 binding observation score and p63 ChIP-seq enrichment in the MCF10A cell line ( A ) or WT CRE activity from the STARR-seq assay ( B ). ( C ) Correlation between WT or mut CRE activity and p63 ChIP-seq enrichment in MCF10A cells (Spearman's ρ=0.127, P = 2.26e-48 for WT comparison and ρ=-0.0, P = 0.98 for mut). ( D ) WT and mut CRE activity for Unique p63RE or p53RE + p63RE motif types with number of each motif type assessed in the experiment indicated on the x -axis (****: P -value < 0.0001, Wilcoxon signed-rank test). ( E ) Relationship between meta-analysis-based p53 observation score and p53 ChIP-seq enrichment in MCF10A cell line. ( F ) Correlation between WT or mut activity and p53 ChIP-seq enrichment in MCF10A cells (Spearman's ρ=0.405, P = 0.0 for WT comparison and ρ=-0.094, P = 4.4e-27 for mut). ( G ) Western blot analysis of p53 and ΔNp63 expression in MCF10A and MCF10A TP53 −/− cells. GAPDH is used as a loading control. ( H ) p53RE + p63RE regulatory element activity in WT or TP53 −/− MCF10A cell lines (****: P -value < 0.0001, Wilcoxon signed-rank test). ( I ) Differences in WT CRE activity between p63RE motif types in MCF10A TP53 −/− cells (****: P -value < 0.0001, Mann–Whitney U test).

Article Snippet: All human mammary epithelial cell lines MCF10A TP53+/+ and TP53 −/− (Sigma-Millipore clls1049) were cultured in 1:1 Dulbecco's Modified Eagle's Medium: Ham's F-12 (Gibco, #11330–032), supplemented with 5% Horse Serum, (Gibco, #16050–122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100–15), 0.5 μg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 μg/mL insulin (Sigma, #I-1882) and 1% penicillin-streptomycin (Gibco, #15240–062).

Techniques: Binding Assay, ChIP-sequencing, Activity Assay, Comparison, Western Blot, Expressing, Control, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity

doi: 10.1093/nar/gkae1143

Figure Lengend Snippet: Analysis of p63RE class and CRE activity. ( A ) PWM of p53RE + p63RE classes, with stars indicating nucleotide substitutions in mut variants. p63 ( B ) or p53 ( C ) ChIP-seq enrichment in MCF10A cells within each p53RE + p63RE class. WT or mut regulatory element activity in MCF10A ( D ) or MCF10A TP53 −/− ( E ) within each p53RE + p63RE class. ( F ) PWM of Unique p63RE classes, with stars indicating nucleotide substitutions in mut variants. ( G ) p63 ChIP-seq enrichment in MCF10A cells within each unique p63RE class. WT or mut regulatory element activity in MCF10A ( H ) or MCF10A TP53 −/− ( I ) based on Unique p63RE motif class. ( J ) DRM frequency in CREs. ( K ) Average GC content across CRE regions separated by motif type. GC content was determined using a 10 nt sliding window approach. Shaded area represents a 95% confidence interval. Average CRE GC content of unique p63RE ( L ) or p53RE + p63RE ( M ) within each motif class. Statistical comparisons were computed using either Mann–Whitney U test ( B, C, G, L, M ) or Wilcoxon signed-rank test ( D, E, H, I ). P -values are indicated as ns : 0.05 < P , **: 0.001 < P ≤ 0.01, ***: 0.0001 < P ≤ 0.001, ****: P ≤ 0.0001.

Article Snippet: All human mammary epithelial cell lines MCF10A TP53+/+ and TP53 −/− (Sigma-Millipore clls1049) were cultured in 1:1 Dulbecco's Modified Eagle's Medium: Ham's F-12 (Gibco, #11330–032), supplemented with 5% Horse Serum, (Gibco, #16050–122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100–15), 0.5 μg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 μg/mL insulin (Sigma, #I-1882) and 1% penicillin-streptomycin (Gibco, #15240–062).

Techniques: Activity Assay, ChIP-sequencing, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity

doi: 10.1093/nar/gkae1143

Figure Lengend Snippet: Characterization of p63-dependent CRE transcriptional activity in MCF10A TP53 −/− cells. p63RE-dependent activity is defined by 1.5 fold-change (WT/mut) cutoff where ‘Activating’ is > 1.5, ‘Repressing’ is < 1.5 and the remaining are defined as ‘Unchanged’. ( A ) Distribution of regulatory element function in MCF10A TP53 −/− cells with each motif type. Distribution of regulatory element function in p53RE + p63RE ( B ) or p63 unique RE ( C ) motif classes as defined in Figure , . ( D ) Average GC content across regulatory element regions based on p63RE-dependent element activity group. Shaded area represents a 95% confidence interval. ( E ) Aggregated regulatory element activity in MCF10A TP53 −/− in each p63RE-dependent activity group (****: P -value < 0.0001, Wilcoxon signed-rank test). Top 30 enriched motifs in ‘Activating’ or ‘Repressing’ regulatory element groups relative to ‘Unchanged’ regulatory element groups. Motif enrichment was performed using HOMER ( , ). Dot size represents the fraction of CREs containing the specified motif. Color scale indicates Bonferroni-corrected inverted P -value. ( G ) Enrichment scores of different TFs from the ReMap 2022 meta-analysis at p63RE-activated (X-axis) or p63RE-repressed (Y-axis) relative to p63RE unchanged background elements. Enrichment scores were calculated using the Giggle genomic interval enrichment approach . ( H ) Reporter gene expression in MCF10A TP53 −/− cells mediated by the selected regulatory elements used in the STARR-seq assay (120 nt). Regulatory element variants included p63RE, AP1 or TEAD motif mutants (****: P -value < 0.0001, One-way ANOVA). Reporter construct schematic is shown at the top. pGL4.24 vector was used to measure minP promoter baseline activity.

Article Snippet: All human mammary epithelial cell lines MCF10A TP53+/+ and TP53 −/− (Sigma-Millipore clls1049) were cultured in 1:1 Dulbecco's Modified Eagle's Medium: Ham's F-12 (Gibco, #11330–032), supplemented with 5% Horse Serum, (Gibco, #16050–122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100–15), 0.5 μg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 μg/mL insulin (Sigma, #I-1882) and 1% penicillin-streptomycin (Gibco, #15240–062).

Techniques: Activity Assay, Gene Expression, Construct, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity

doi: 10.1093/nar/gkae1143

Figure Lengend Snippet: Effect of p63 isoforms on p63-dependent CRE activity. ( A ) Western blot analysis of p63 isoform expression in HCT116 TP53 −/− cells. ( B ) p63RE-dependent reporter gene expression ( SFN- derived BDS2,3, ) in response to four different transiently expressed p63 isoforms, and an empty pcDNA3.1 vector as a negative control, in HCT116 TP53 −/− cells 24 h after transfection (****: P -value < 0.0001, Two-way ANOVA). ( C ) Western blot showing Doxycycline-inducible TAp63β or GUS control expression in MCF10A cells. Cells were treated for 8 h with either 500 ng/ml Doxycycline (Dox) or water as vehicle control. ( D ) WT and mut CRE activity ( n = 13 532) in either control (GUS) or TAp63β induced cell line (****: P -value < 0.0001, Wilcoxon signed-rank test). ( E ) Distribution of p63-dependent CRE activity in either GUS (control) or TAp63β overexpressing cell line.

Article Snippet: All human mammary epithelial cell lines MCF10A TP53+/+ and TP53 −/− (Sigma-Millipore clls1049) were cultured in 1:1 Dulbecco's Modified Eagle's Medium: Ham's F-12 (Gibco, #11330–032), supplemented with 5% Horse Serum, (Gibco, #16050–122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100–15), 0.5 μg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 μg/mL insulin (Sigma, #I-1882) and 1% penicillin-streptomycin (Gibco, #15240–062).

Techniques: Activity Assay, Western Blot, Expressing, Gene Expression, Derivative Assay, Plasmid Preparation, Negative Control, Transfection, Control