Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: DOG screening overview. a , Screening overview showing the heatmaps (average of n = 3) of the cell proliferation at 24 h and 72 h for both the MCF10a and MCF7 cells quantified as Ki-67 positive nuclei. The heatmaps corresponding to the cell density and cell area can be found in the supplementary figures. b , Scatter plots showing all data points from one screening (4 DOGs) ranked from low to high. Tissue culture polystyrene (TCP) was used as a control. The 10 % lowest and 10 % highest scoring datapoints are shown on the side and are shown to be significantly different (p < 0.0001). Annotation of black dot with “T” or “L” corresponds to the representative fluorescence images of “Top” and “Low” hits as shown in .

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques: Control, Fluorescence

Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: Top- and low-hit Ki-67-expressing proliferative cells after 72 h of cell culture . Cells were stained for Ki-67 (red), phalloidin (green), and nuclei (blue). Scale bar is 100 μm. White arrows show examples of Ki-67 positive nuclei. The underlying material properties of each image are: MCF10a top hit: S-W 51° 251 MPa, MCF10a low hit: T-S|W 3.8 μm 82° 47 MPa, MCF7 top hit: S-W 63° 42 MPa, MCF7 low hit: T-SW 6 μm 68° 63 MPa.

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques: Expressing, Cell Culture, Staining

Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: Scatter plots comparing the effect of each individual physicochemical parameter of the DOG between MCF10a and MCF7 cells. The material parameters (topography, wettability, and stiffness on the same data points, shown with the corresponding color scale) were shown on the biological output (cell proliferation, % Ki-67 positive cells) for the MCF10a vs. the MCF7 cells. Similar plots showing the scatter plots for cell density and cell area can be found in .

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques:

Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: ROIs were chosen that lead to a higher proliferation after 72 h for MCF10a than MCF7 cells. a , Regions of interest (ROIs) are chosen as points furthest away from the dashed line. In orange and blue, points are indicated that show an individual statistical difference (P < 0.05) when comparing the proliferation percentage of MCF7 vs. MCF10a. Orange or blue dots indicate an increase for either MCF7 or MCF10a proliferation, respectively. b , Points from a were grouped and show a significant different in both MCF10a and MCF7 Ki-67 positive cells percentage (p < 0.0001). c , Individual DOG values for each ROI chosen in a are shown. Corresponding fluorescence images for each ROI can be found in .

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques: Fluorescence

Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: ROIs were chosen from the screening as “positive” when MCF10a Ki-67 % > MCF7 Ki-67 % (labelled as Pos1, Pos2, Pos3, shown in blue) and “negative” when MCF7 Ki-67 % > MCF10a Ki-67 % (labelled as Neg1, Neg2, Neg3, shown in orange). a , Cell experiments were repeated on translational (labelled as Trans) substrates with those specific material properties and compared to the proliferation (% Ki-67 positive cells after 72 h of cell culture) rates from the screening (Screen). b , Cell density after 72 h was compared between the screening and translation findings. c , Fluorescence images of two example ROIs, Pos1 and Neg1. Cells were stained for Ki-67 (red), phalloidin (green), and DAPI (blue). Corresponding fluorescence images for the other ROIs can be found in . The scale bar is 100 μm.

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques: Cell Culture, Fluorescence, Staining

Journal: Bioactive Materials

Article Title: Harnessing the power of physicochemical material property screening to direct breast epithelial and breast cancer cells

doi: 10.1016/j.bioactmat.2025.04.003

Figure Lengend Snippet: Proliferation quantified as % Ki-67 positive cells after 72 h for both MCF7 and MCF10a cells compared between the translational cell experiments on translated ROIs for both low (shown in blue, 1000 cells cm −2 ) and high (shown in orange, 5000 cells cm −2 ) seeding density. a , Individual values for each ROI compared between low and high seeding density. b , All low and high seeding density datapoints from a were grouped and compared for statistical difference (n.s.: p > 0.05, ∗: p < 0.05).

Article Snippet: Normal breast epithelial cells (MCF10a, Aldrich) were maintained in MCF10a Complete Medium (EP-ML-0525, Elabscience) containing 5 % horse serum, 20 ng mL −1 epidermal growth factor (EGF), 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1 % non-essential amino acid solution (NEAA), and 1 % penicillin/streptomycin.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Suppression of cancer cell COX-2 expression by soluble factors from fibroblasts. (A) A549 lung cancer cells (106 cells) were cocultured with HsFb at various cell densities in a Boyden chamber for 24 h. A549 cells were removed and treated with PMA (100 nM) for 4 h. COX-2 proteins in the cell lysates were analyzed by Western blotting. (B) Conditioned medium (CM) was collected from Hs68 fibroblasts (HsFb) or A549 cells cultured in medium containing 2.5% FBS for 24 h. (Left) HsFb CM was added to washed A549 cells. (Right) A549 CM was added to washed serum-starved Hs68 fibroblasts. Cells were treated with PMA for 4 h, and COX-2 proteins were analyzed by Western blotting. Error bars denote mean ± SEM (n = 3). (C) CMF2 fractions prepared from HsFb or A549 CM were added to washed A549, HT29, or serum-starved HsFb, and PMA-induced COX-2 was analyzed. CMF2 of HsFb inhibited COX-2 expression whereas CMF2 of A549 had no effect. Two independent experiments were performed with similar results. (D) Representative mass spectra (m/z 0–300) of CMF2 prepared from HsFb or A549. Control denotes F2 fraction of the basal DMEM containing 2.5% FBS. Metabolomic analysis of CMF2 was performed using UPLC–QTof mass spectrometry. (E) Comparison of relative intensity of m/z 276.1 and m/z 262.1 between HsFb CMF2 and A549 CMF2. Error bars denote mean ± SEM (n = 3). (F) Effect of cancer-associated fibroblasts on PMA-induced COX-2 expression in A549 cells. CM and CMF2 prepared from prostate cancer-associated fibroblasts (PcFb) inhibited COX-2 expression whereas neither CM nor CMF2 of breast cancer-associated fibroblasts (bCAF) did. (G and H) The metabolomic profile of (G) PcFb-CMF2 was similar to that of HsFb-CMF2, whereas the profile of (H) bCAF-CMF2 reveals diminished m/z 276.1, m/z 262.1, and other m/z peaks.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Expressing, Western Blot, Cell Culture, Control, Mass Spectrometry, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Tryptophan hydroxylase (TPH-1)-derived 5-HTP suppresses COX-2 expression. (A) TPH-1 proteins in HsFb vs. A549 cells were analyzed by Western blotting. The error bars refer to mean ± SEM (n = 3). (B) TPH-1 protein levels in siRNA-transfected HsFb vs. control scRNA-transfected HsFb. “Basal” denotes untransfected cells. (C) Analysis of COX-2 proteins in HsFb transfected with TPH-1 siRNA or scRNA. COX-2 was increased in siRNA-treated cells, and COX-2 suppression was restored by 5-HTP (10 μM) or 5-MTP (10 μM) supplement. 5-MTP supplement completely inhibited COX-2 expression whereas 5-HTP partially suppressed COX-2 proteins. (D) HsFb were pretreated with 5-HTP followed by PMA for 4 h. COX-2 proteins were analyzed by Western blotting. (E) Metabolomic analysis of CMF2 prepared from CM of HsFb with or without siRNA or scRNA transfection.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Endogenous and exogenously added 5-MTP inhibits COX-2 expression. (A) Pure 5-MTP at increasing concentrations was added to A549 treated with or without PMA for 4 h. COX-2 proteins were analyzed by Western blotting. (B) 5-MTP restored COX-2 suppression in TPH-1 siRNA-transfected HsFb in a concentration-dependent manner. (C) Addition of 5-MTP to A549 cells enhances m/z 276.1 and 262.1 peaks on mass spectrometry. (D) HsFbs were transfected with TPH-1 siRNA or scRNA. l-Tryptophan (Try) or 5-HTP was added to the transfected cells. Conditioned medium was collected, and 5-MTP concentration was measured by EIA. Each bar denotes mean ± SEM (n = 3). ns, not statistically significant. (E) Analysis of COX-2 proteins by Western blotting in HsFb transfected with HIOMT siRNA or a control scRNA. (Upper) Representative blot. (Lower) Quantitative analysis by densitometry. Error bars denote mean ± SEM (n = 3). Concentration of 5-MTP was 10 μM. (F) Measurement by EIA of 5-MTP in the CM of HsFb under the indicated treatment; 10 μM of 5-HTP or 5-MTP was added. Each error bar denotes mean ± SEM of three independent experiments done in triplicate. ns, not statistically significant.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Expressing, Western Blot, Transfection, Concentration Assay, Mass Spectrometry, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: 5-MTP rescues A549 defects in 5-MTP synthesis and cell proliferation and migration. (A) Analysis of HIOMT proteins in A549 vs. HsFb. (Upper) Representative blot. (Lower) Densitometric analysis of three independent experiments. The error bars are mean ± SEM (*P < 0.01). (B) Measurement of 5-MTP level in A549 vs. HsFb CM. Error bars are mean ± SEM (n = 3). (C) Neither Try nor 5-HTP increased A549 5-MTP production, whereas addition of exogenous 5-MTP increased the 5-MTP level measured by EIA. The error bars are mean ± SEM (n = 3). (D) A549 cells labeled with MTT were treated with 5-MTP for 4 or 24 h. Each bar denotes mean ± SEM (n = 3). (E) A549 cells were pretreated with 5-MTP (10 μM) for 30 min followed by PMA (100 nM) for 6 or 24 h. (Upper panels) Representative cell migration. (Lower panels) Migrated cell counts expressed as percentage of basal controls. Error bars denote mean ± SEM (n = 3). (F) A549 cells were seeded on regular or matrigel-coated membrane and pretreated with 5-MTP for 30 min followed by PMA. (Upper) Representative cell migration and invasion. (Lower panels) Quantitative analysis of migration and invasion.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Migration, Labeling, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: 5-MTP reduces cancer growth and metastasis in a murine tumor xenograft model. A549 (5 × 106 cells) were transfected with luciferase (A549-luc-c8) and injected s.c. into the flank of SCID-Beige mice. Ten mice each received i.p. injection of 5-MTP (100 mg/kg) or vehicle twice weekly. (A) Caliper measurement of the s.c. tumor volume periodically for 7 wk. *P < 0.05 and **P < 0.01. (B) Tumor growth was monitored by in vivo bioluminescent imaging (IVIS). The time courses of IVIS of two representative vehicle-treated and two representative 5-MTP–treated mice are illustrated. (C) 5-MTP reduces lung metastasis. Mice were euthanized and both lungs were removed. Nodules were counted. Each nodule was sectioned and stained with hematoxylin-eosine and examined under the microscope. 5-MTP pretreatment significantly attenuated lung metastasis. (D) Metabolic scheme illustrating 5-MTP biosynthesis in HsFb vs. A549 cancer cells. The dotted line denotes possible defects in the synthetic pathway.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Transfection, Luciferase, Injection, In Vivo, Imaging, Staining, Microscopy

Journal: Oncology Letters

Article Title: Expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells

doi: 10.3892/ol.2013.1703

Figure Lengend Snippet: Expression and location of TRAF4 in breast cancer cells. (A) Results of immunofluorescence staining showing TRAF4 (green fluorescence) localized in the cytoplasm and nuclei (scale bar, 20 μm). (B) MCF-10A, MCF-7 and MDA-MB-231 cells were lysed and the lysates were subjected to western blot analyses of TRAF4 and β-actin. Results showed that the total expression of TRAF4 in the MCF-10A cells was lower than in the MCF-7 and MDA-MB-231 cells ( ** P=0.002 and P=0.001, respectively). TRAF4, tumor necrosis factor receptor-associated factor 4; IDV, integated optical density value.

Article Snippet: The membranes for western blotting were incubated with mouse anti-human monoclonal antibodies against TRAF4 (1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), NF-κB p65 (1:1,000; Beyotime Institute of Biotechnology, Haimen, China), lamin B1 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and β-actin (1:1,000; Santa Cruz Biotechnology Inc.), followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Oncology Letters

Article Title: Expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells

doi: 10.3892/ol.2013.1703

Figure Lengend Snippet: TRAF4 suppresses the activation of NF-κB in MCF-7 Cells. The MCF-7 cells were lysed and the lysates were subjected to western blot analyses of NF-κB, β-actin and lamin B1. Following TNF-α treatment (10 ng/ml; 15 min), the nuclear expression of NF-κB was significantly downregulated when TRAF4 was knocked down in the MCF-7 cells ( ** P=0.002). TRAF4, tumor necrosis factor receptor-associated factor 4; NF-κB, nuclear factor κB.

Article Snippet: The membranes for western blotting were incubated with mouse anti-human monoclonal antibodies against TRAF4 (1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), NF-κB p65 (1:1,000; Beyotime Institute of Biotechnology, Haimen, China), lamin B1 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and β-actin (1:1,000; Santa Cruz Biotechnology Inc.), followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Oncology Letters

Article Title: Expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells

doi: 10.3892/ol.2013.1703

Figure Lengend Snippet: Biological function of TRAF4 in MCF-7 cells. (A) MTT results showing that cell proliferation was significantly suppressed following the knockdown of TRAF4 ( * P=0.02). Results of flow cytometry showed that TRAF4 ablation resulted in (B) significant increase of G 1 phase cells ( ** P=0.009) and reduction of S phase cells ( * P=0.04) and (C) significant increase in the early apoptotic cells ( ** P=0.001). All comparisons were made between the groups of MCF-7 cells or cells transfected with negative control alone. TRAF4, tumor necrosis factor receptor-associated factor 4.

Article Snippet: The membranes for western blotting were incubated with mouse anti-human monoclonal antibodies against TRAF4 (1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), NF-κB p65 (1:1,000; Beyotime Institute of Biotechnology, Haimen, China), lamin B1 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and β-actin (1:1,000; Santa Cruz Biotechnology Inc.), followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Flow Cytometry, Transfection, Negative Control

Journal: Nature Communications

Article Title: Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation

doi: 10.1038/s41467-017-00116-3

Figure Lengend Snippet: UNC119-solubilising activity maintains SFK PM localisation. Confocal micrographs of MCF10a cells expressing Src-mCit ( a ) or Fyn-mCit ( b ) and the ER marker, CalR-mCer transfected with UNC119 targeting siRNA ( lower rows ) or non-targeting siRNA control nucleotides ( upper rows ). c Representative western blot showing UNC119 levels of transfected cells and the GAPDH-loading control. Bar graph depicts the quantification of UNC119 kD in which UNC119 levels were normalised to the tubulin-loading control and the non-targeting siRNA control ( n = 4, data are mean ± SD; significance determined by Student’s t -test). d Quantification of co-localisation with Mander’s coefficient for SFK-mCit with the ER CalR-mCer marker in MCF10a cells ( n > 30 cells per condition from two independent experiments; data are mean ± SD. * P < 0.05; *** P < 0.001; Student’s t -test). Confocal micrographs of HeLa cells expressing Src-mCit e or Fyn-mCit f and the ER marker; CalR-mCer e or stained with anti-Calnexin antibodies f , transfected with UNC119 targeting siRNA ( lower rows ) or non-targeting siRNA control nucleotides ( upper rows ). g Quantification of co-localisation with Mander’s coefficient for SFK-mCit with the ER markers for HeLa cells ( n > 15 cells per condition from two independent experiments; data are mean ± SD. * P < 0.05; *** P < 0.001; Student’s t -test). h Quantification of UNC119 KD in HeLa cells by western blot using UNC119 and tubulin antibodies. UNC119 levels were normalised to the tubulin-loading control and the non-targeting siRNA control ( n = 5, data are mean ± SD; ** P < 0.001; Student’s t -test). i Confocal micrographs of MCF10a cells transfected with UNC119A and UNC119B targeting siRNA or non-targeting siRNA control nucleotides and then stained with an antibody against SFKs ( green ) and with DAPI ( blue ). Traces below show the vertical intensity profile plots of SFK for the yellow dashed area . j The mean SFK intensity profile plots for multiple cells ( n = 20 cells per condition, mean intensity ± SEM). Scale bars , 10 μm

Article Snippet: MCF10a cells (CRL-10317; LGC Genomics GmbH, Berlin, Germany) were cultured in a DMEM/F12 culture medium (PAN-Biotech GmbH) supplemented with glutamine, 5% horse serum (PAN-Biotech GmbH), 20 ng/ml epidermal growth factor (Sigma-Aldrich Chemie GmbH, Munich, Germany), 0.5 mg/ml hydrocortisone (Sigma-Aldrich Chemie GmbH), 100 ng/ml cholera toxin (Sigma-Aldrich Chemie GmbH) and 10 µg/ml insulin (Sigma-Aldrich Chemie GmbH).

Techniques: Activity Assay, Expressing, Marker, Transfection, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Venn diagram indicating differentially up-regulated genes (Nutlin-3A/DMSO, fold-change ≥2, adjusted p < 0.05) in MCF10A or SkFib cells. B, gene ontology analysis of differentially expressed genes from MCF10A and SkFib cells showing up-regulated genes for the top three significant biological processes. B–D, qRT-PCR validation of two representative common (C), MCF10A-specific (D), and SkFib-specific (E) Nutlin-3A–induced targets. Target gene expression is normalized to GAPDH (relative expression: Rel. Exp.), and data represent technical replicates from three independent biological replicates. Error bars represent S.E. with p values calculated by Student's t test, ****, p < 0.0001. F, intersection between SkFib (top) or MCF10A (bottom) p53 targets identified in this work compared with the meta-analysis of p53 targets identified in at least three independent experiments (32). G, immunoblotting for p53 expression at 6 h of DMSO (D) or Nutlin-3A (N) treatment in SkFib cells stably expressing shRNA against p53 (p53sh) or a nontargeting control shRNA (scr). H, qRT-PCR analysis of p53 expression at 6 h of DMSO (D) or Nutlin-3A (N) treatment in SkFib cells stably expressing shRNA against p53 or a nontargeting control shRNA. p53 expression is normalized to GAPDH expression (relative expression). Error bars represent S.E.; ****, p < 0.0001, and ***, p < 0.001, calculated by Student's t test. I, qRT-PCR analysis of SkFib-specific gene targets, GDNF and TRIM55, in SkFib cells stably expressing an shRNA to p53 or a nontargeting control shRNA. Gene expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001; ***, p < 0.001; and **, p < 0.01, calculated by Student's t test. J, immunoblotting for p53 expression at 6 h of DMSO or Nutlin-3A treatment in response to p53 knockdown in MCF10A cells stably expressing shRNA against p53 or a nontargeting control shRNA. K, qRT-PCR analysis of p53 expression at 6 h of DMSO or Nutlin-3A treatment in response to p53 knockdown in MCF10A cells stably expressing shRNA against p53 or a nontargeting control shRNA. p53 RNA expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, calculated by Student's t test. L, qRT-PCR analysis of MCF10A-specific Nutlin-3A–induced genes, RIC3 and IL1B, in MCF10A cells stably expressing an shRNA to p53 (p53 sh) or a nontargeting control shRNA. Expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, calculated by Student's t test.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Targeted Gene Expression, Expressing, Western Blot, Stable Transfection, shRNA, Control, Gene Expression, Knockdown, RNA Expression

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Venn diagram depicting overlap between significantly-enriched (p < 0. 01, MACS version 2) Nutlin-3A–induced p53 peaks in MCF10A or SkFib. B, boxplots depicting enrichment of p53 (input-normalized, log2) at common p53-binding sites found in both MCF10A (blue, left) and SkFib (green, right). C, fold-change ratio of Nutlin-3A/DMSO of common input-subtracted p53 enrichment for MCF10A or SkFib. D and E, boxplot analysis of the input-subtracted p53 enrichment for MCF10A (blue, left) or SkFib (green, right) for MCF10A-specific (D) or SkFib-specific p53-binding sites (right) (E) in response to DMSO (D) or Nutlin-3A (N) treatment. F–H, Chip-qPCR validation of the common (F), skin fibroblast–specific (G), and MCF10A-specific (H) p53 target CDKN1A/p21 at p53-binding and -nonbinding sites. Genome browser view of replicate ChIP-seq data for MCF10A (blue) and SkFib (green) with the location of qPCR primers for p53-binding site and negative region are shown above the qPCR data. qPCR data represent three biological replicates in MCF10A and SkFib cells with either control (scr, nontargeting) or p53-targeting shRNA after 6 h of Nutlin-3A or DMSO treatment. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001; ***, p < 0.001; and **, p < 0.01.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay, ChIP-qPCR, Biomarker Discovery, ChIP-sequencing, Control, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, representative UCSC Genome Browser track view of GDNF locus, a fibroblast-specific p53 target, in response to DMSO (D) or Nutlin-3A (N) treatment. p53-bound, putative enhancers (H3K27ac+, H3K4me2+, and H3K4me3−) illustrated by a dashed box for GDNF and TRIM55, and the putative GDNF promoter (H3K27ac/H3K4me2/H3K4me3+) is represented by a solid box. MCF10A-N-1 is biological replicate of MCF10A-N-2, and SkFib-N-1 is a biological replicate of SkFib-N-2. The y axis is scaled to the maximum intensity between MCF10A or SkFib for each feature. B, representative UCSC Genome Browser track view at the RIC3 locus, illustrating p53 binding to an MCF10A-specific enhancer signature (H3K27ac/H3K4me2+ and H3K4me3−) in response to Nutlin-3A treatment (dashed box). MCF10A-N-1 is a biological replicate of MCF10A-N-2, and SkFib-N-1 is a biological replicate of SkFib-N-2. The y axis is scaled to the maximum intensity between MCF10A or SkFib for each feature.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, p53 ChIP-seq enrichment from either MCF10A (blue) or SkFib (green) at MCF10A-specific p53-binding sites. (−/+ 2000 bp from the p53 motif; −2 indicates 2000 bp downstream from the motif; 0 indicates the center of the p53 DNA motif, and +2 indicates 2000 bp upstream of the motif.) Two biological replicates for each dataset are shown. H3K4me2 (B) and H3K27ac (C) enrichment in DMSO or Nutlin-3A–treated MCF10A or SkFib within a 4000-bp window (−/+ 2000 bp from the p53 motif; 2 indicates 2000 bp downstream from the motif, 0 indicates the motif center, and +2 indicates 2000 bp upstream from the motif). D–F, percentage of intersecting H3K27ac peaks (input-normalized, MACS version 2, p < 0.01) with p53 peaks (input-normalized, MACS version 2, p < 0.01) that are common to MCF10A and SkFib (D), MCF10A-specific (E), or SkFib-specific (F) in response to DMSO (D) or Nutlin-3A (N) treatment. Adjacent boxplots depict H3K27ac enrichment over a 500-bp window from the p53-binding site.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Homer-derived transcription factor motif enrichment found in MCF10A-specific (top) or SkFib-specific (bottom) enhancers (H3K4me2+/H3K27ac+/H3K4me3−). Full list of transcription factor enrichment and facet-specific enhancers are found in Table S4. B, qRT-PCR analysis of p53, p63, or p73 of cell lysates from MCF10A, SkFib, HUVEC, and HaCaT cells at 6 h after DMSO (D) or Nutlin-3A (N) treatment. Expressions detected by qRT-PCR are normalized to GAPDH expression. HaCaT cells were used as positive control for p63 and p73 expression. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001. C, qRT-PCR analysis of TA–p63 and ΔN-p63 of cell lysates from MCF10A, SkFib, and HCT116-TA–p63 cells. Transiently transfected HCT116 cells with TA–p63 were used as a positive control for TA–p63 expression. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001. D, immunoblotting for p63, TA–p63, and ΔN–p63 of cell lysates from HCT116, HCT116-TAp63, and HCT116-ΔNp63 transiently transfected cells to confirm antibody specificity for different isoforms. E, immunoblotting for p63, TA–p63, or ΔN-p63 in cell lysates from MCF10A cells. HCT116 cell lysate is used as negative control. F, representative UCSC Genome Browser track view of the IL1B locus, illustrating three MCF10A-specific putative enhancers bound by p53 and p63 in response to Nutlin-3A treatment (H3K27ac+, H3K4me2+, H3K4me3−; dashed box). The y axis is scaled to the maximum intensity for each data set. MCF10A-D-1 is a biological replicate of MCF10A-D-2; MCF10A-N-1 is a biological replicate of MCF10A-N-2; and SkFib-N-1 is a biological replicate of SkFib-N-2. G, representative UCSC Genome Browser track view of the RNASE7 locus, illustrating a MCF10A-specific putative enhancer bound by p53 and p63 in response to DMSO (D) or Nutlin-3A (N) treatment (H3K27Ac+, H3K4me2+, H3K4me3−; dashed box). The y axis is scaled to the maximum intensity for each data set. MCF10A-D-1 is a biological replicate of MCF10A-D-2; MCF10A-N-1 is a biological replicate of MCF10A-N-2; and SkFib-N-1 is a biological replicate of SkFib-N-2. H, Venn diagram representation of overlapping p53 and p63 ChIP-seq peaks (input-normalized, p53/p63 motif-positive, MACS version 2, p < 0.01) in MCF10A when treated with Nutlin-3A. I, heatmap plots of p53 and p63 enrichment at shared binding sites in replicate MCF10A cells within a 2000-bp window (−/+ 1000 bp from the peak center) in response to Nutlin-3A treatment. J, percentage of intersecting H3K27ac/H3K4me2+ peaks (input-normalized, MACS version 2, p < 0.01) with p53 only, p63 only, or p53/p63 peaks observed in MCF10A cells (input-normalized, MACS version 2, p < 0.01). K, percentage of p63-binding sites observed in MCF10A cells at varying distances to the nearest TSS of all RefSeq genes (white) or Nutlin-3A–induced genes (red).

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Positive Control, Transfection, Western Blot, Negative Control, ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, qRT-PCR analysis of p53 and p63 in response to p63 knockdown in MCF10A cells stably expressing shRNA to p63 or a nontargeting control shRNA (scr) after 6 h of DMSO (D) or Nutlin-3A (N) treatment. Target gene expression is normalized to GAPDH for qRT-PCR analysis. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001, and ***, p < 0.001. B, immunoblotting for p53, p63, and GAPDH in MCF10A cells stably expressing shRNA to p53, p63, or a nontargeting control shRNA after 6 h of DMSO or Nutlin-3A treatment. This immunoblot image is an uncropped version of Fig. 1J. C, RNA-seq analysis of MCF10A-specific, Nutlin-3A–induced genes in MCF10A cells expressing shRNA targeting either a nontargeting control, p53, or p63. Data are graphed as fold-change (Nutlin-3A/DMSO, log2, median in black). Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001; ***, p < 0.001; and *, p < 0.05. D, qRT-PCR analysis of MCF10A-specific Nutlin-3A–induced genes, RIC3, IL1A, and IL1B. Expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, and **, p < 0.01, calculated by Student's t test. E, percent of p53, p63, and p53/p63 co-bound sites relative to the three classes of p63-regulated p53 gene targets (p63-dependent, p63-independent, and p63-inhibited) in binned distance regions (under 25 kb and over 25 kb). Statistics represent χ2 test using the distance of each binding site class to nonregulated genes.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Quantitative RT-PCR, Knockdown, Stable Transfection, Expressing, shRNA, Control, Targeted Gene Expression, Western Blot, RNA Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, H3K4me2 enrichment (input-subtracted H3K4me2, −/+ 250 bp from p53 motif center) at p53-binding sites in MCF10A cells expressing control (scr), p53, or p63-targeted shRNA in response to DMSO or Nutlin-3A treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001. B, H3K27ac enrichment at p53-binding sites in MCF10A cells expressing the represented shRNA molecules after 6 h of DMSO (D) or 5 μm Nutlin-3A (N) treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001, and ***, p < 0.001. C, bar graph displaying the number of p53-binding sites (left) or total cellular complement of H3K27ac+/H3K4me2+/H3K4me3− enhancers with more than 2-fold change in H3K27ac enrichment after Nutlin-3A treatment of MCF10 cells expressing the indicated shRNA. D, H3K4me2 enrichment at p53-binding sites (25) in HCT116 TP53+/+ or −/− cells in response to DMSO or Nutlin-3A treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001, and **, p < 0.01. E, H3K4me1 and H3K4me2 enrichment at p53-binding sites (85) in Trp53+/+ or Trp53−/− mouse embryonic fibroblasts.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay, Expressing, Control, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A and B, input-subtracted H327ac (A) or H3K4me2 (B) enrichment at p63-binding sites (left, −/+ 250 bp from p63 peak center) or at all remaining enhancers (H3K27ac+, H3K4me2+, H3K4me3−, right) in MCF10A cells expressing nontargeting (Scr) or p63 shRNA. C, representative UCSC Genome Browser track view of the RRM1 locus, illustrating three MCF10A-specific putative enhancers bound by p63 that are lost in response to p63 depletion (H3K27ac+, H3K4me2+, H3K4me3−; dashed box). D, representative UCSC Genome Browser track view of the EDN2 locus, illustrating three MCF10A-specific putative enhancers bound by p63 that are lost in response to p63 depletion (H3K27ac+, H3K4me2+, H3K4me3−; three separate dashed boxes). The y axis is scaled to the maximum intensity for each data set. E, bar graphs depicting the percent of p63 peaks that show 2-fold gains or losses of H3K27ac (left) or H3K4me2 (right) in response to either p53 or p63 depletion relative to nontargeting control shRNA. F, number of p63-sensitive, H3K4me2-marked enhancers (out of 1496 total) overlapping DHS across epithelial and nonepithelial cell types analyzed by the ENCODE project. Error bars represent the median and 95% confidence interval. G, Jitter plot depicting the fraction of p63-dependent or -independent enhancers overlapping regions of DHS across both epithelial and nonepithelial cell lines as assayed by the ENCODE project. H, heatmap of k-means (k = 3) clustered Bonferroni-corrected p values (q-values) for motif enrichment found at p63-dependent or -independent enhancers. Blue represents adjusted p values less than 0.05, and white represents adjusted p values greater than 0.05. A full list of motifs and their enrichment statistics can be found in Table S7.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay, Expressing, shRNA, Control