Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: Experimental modulation of EP300 in cellular models. Expression of EP300 and E-cadherin was determined by immunoblot analyses. a EP300 was downregulated in breast cancer luminal MCF-7 cells by lentiviral expression of two different EP300 hairpins (MCF7-shEP300-1 and MCF7-shEP300-2). Cells expressing the empty pGIPZ vector (MCF7-shev) were used as control. b , a genetic knock-out of EP300 (HCT-KOEP300) is available in colon carcinoma HCT116 cells. This cell line is hemizygous for the EP300 locus and generates a C-terminus truncated EP300 protein —note its lower molecular mass (tEP300, truncated EP300). Paclitaxel-resistant derivatives are indicated with the -TX name extension. c , d EP300 was upregulated in breast cancer basal-like CAL51 and MDA-MB-231 cells with an EP300 expression cassette in pcDNA3.1 (CAL-EP300 and MDA-EP300). In both cases, cells transfected with pcDNA3.1 were used as controls (CAL-ev and MDA-ev). Lamin B was used as a loading control. Representative pictures of three replicates are shown. Immunoblots were quantified and data are shown in the histograms as average ± SD of three blots. All statistical comparisons (* P < 0.05) versus control cells

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Expressing, Western Blot, Plasmid Preparation, Control, Knock-Out, Transfection

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: Modulation of paclitaxel sensitivity after experimental modulation of EP300 expression in cell models

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Expressing

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: EP300 regulates the generation of paclitaxel resistance. Cells were treated for 3 days with paclitaxel and drug-resistant clones were stained with crystal violet 3 weeks after ( left panels ) and counted ( right panels ). a , MCF-7 cells. b , HCT116 cells. c , CAL51 cells. Numerical data represent the average ± SD of three independent experiments. All statistical comparisons (* P < 0.05) versus control cells. Pictorial data show a representative of three different experiments

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Clone Assay, Staining, Control

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: EP300 regulates stem cell markers. a – c Flow cytometry plots after staining ( a MCF-7 cells; b HCT116 cells; c MDA-MB-231 cells) with CD44-APC- and CD24-PE-conjugated antibodies. Paclitaxel-resistant cell derivatives (MCF7-shEP300-1-TX, MCF7-shEP300-2-TX and HCT-KOEP300) were generated from the corresponding cells after selection with 20 nM (MCF-7 cells) and 40 nM paclitaxel (HCT116 cells). Histograms indicate the percentage of CD44 + /CD24 − cells. d Upregulation of EP300 in CAL51 cells reduces the percentage of ALDH + cells. Cells were treated with Aldefluor alone ( ALDH ) or in the presence of the ALDH inhibitor diethylaminobenzaldehyde ( Control ) and then analysed by flow cytometry. The green gate was set up with the control cells to include no more than 1% of the population and was used to determine the percentage of ALDH-positive cells in the absence of inhibitor. Representative flow cytometry plots are shown. Numerical data represent the average ± SD of three independent experiments. All statistical comparisons (* P < 0.05) versus control cells

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Flow Cytometry, Staining, Generated, Selection, Control

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: EP300 regulates anchorage independence. Anchorage independence was determined by mammosphere formation in low attachment plates ( a – c ) and the formation of clones in soft agar ( d – e ) after 2 and 4 weeks, respectively. Representative pictures of at least three independent experiments are shown. Numerical data indicate the anchorage independence efficiency after counting tumour spheres larger than 50 µm in diameter and is represented as the average ± SD of three independent experiments. All statistical comparisons (* P < 0.05) versus control cells

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Clone Assay, Control

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: Genome-wide expression profile of EP300-downregulated MCF-7 cells, and their paclitaxel-resistant derivatives. a Hierarchical clustering of differentially expressed genes. Differentially upregulated expression values are shown in red , downregulated in green . Scale represents colour values corresponding to lg 2 expression. b , Venn diagram indicating the number of differentially expressed genes in each of the pair-wise comparisons to MCF7-shev control cells. There were 4044 common differentially expressed genes present in all cells after downregulation of EP300. c Eleven genes were selected for validation by quantitative PCR. The top panel for each gene shows the normalized fluorescence from Affymetrix array expression data. The lower panel for each gene indicates the normalized QPCR data relative to the expression data obtained in control MCF7-shev cells. QPCR data represent the mean ± SD from three replicates

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Genome Wide, Expressing, Control, Biomarker Discovery, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: Validation of the EP300 signature in other cell models. a QPCR data from EP-downregulated minimally transformed mammary epithelial cells, and their paclitaxel-resistant derivatives. The gene set is the same that is used in Fig. c to validate the MCF-7 signature. b QPCR data of anti-apoptotic BCL2 and stem cell marker ABCG2 from the MCF-7 signature in HCT116 cells. c and d , Bcl-2 immunoblot ( top panels ) and blot quantification ( lower panels ) in both MCF-7 ( c ) and HCT116 ( d ) cell derivatives. β-actin is used as a loading control. Numerical data represent the average ± SD of three independent experiments. All statistical comparisons (* P < 0.05) versus control cells. Immunoblot shows a representative of three independent experiments

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Biomarker Discovery, Transformation Assay, Marker, Western Blot, Control

Journal: Breast Cancer Research and Treatment

Article Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

doi: 10.1007/s10549-017-4202-z

Figure Lengend Snippet: EP300 is downregulated in metaplastic breast cancer. a Validation of EP300 antibody using 10- and 100-fold molar excess competing peptide in two independent breast cancer samples. H&E, haematoxylin and eosin staining. Scale bar , 200 μm. b EP300 and E-cadherin staining in three representative samples of normal breast and metaplastic breast cancer. H&E, haematoxylin and eosin staining. Scale bar , 200 μm. c Loss of EP300 nuclear staining in metaplastic breast cancer. Pictures show representative zoomed-in shots illustrating nuclear EP300 localization in normal breast and its loss in metaplastic breast cancer. Scale bar , 20 μm. d Representative EP300 and E-cadherin staining in a metaplastic breast cancer sample with a squamous epithelium nest (bottom half). The top half is composed of spindle-like cells. Note the absence of E-cadherin and EP300 expression in the mesenchymal component but positive staining in the squamous nest. Scale bar , 200 μm

Article Snippet: EP300 antibody specificity was validated with a synthetic peptide (APrEST73567, Atlas Antibodies) competition assay following manufacturer’s protocol.

Techniques: Biomarker Discovery, Staining, Expressing

Journal: Scientific Reports

Article Title: Curcumin inhibits glycolysis via EP300 in oral squamous cell carcinoma

doi: 10.1038/s41598-026-44496-3

Figure Lengend Snippet: Role of EP300 in Curcumin-Mediated Suppression of Glycolysis in Oral Squamous Cell Carcinoma. ( A )Validation of EP300 overexpression in UM-SCC-1 cells. Relative EP300 mRNA (left) and protein levels (right) were assessed by qRT-PCR and Western blot, respectively. Compared with negative control (NC) and wild-type (WT) cells, EP300-oe cells showed markedly upregulated EP300 expression at both the mRNA and protein levels. ( B ) Comparison of EP300 mRNA and protein expression levels among different treatment groups. ( C ) Quantitative analysis of glucose uptake capacity across groups. ( D ) Quantitative analysis of lactate release capacity across groups. Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns = not. The negative control (NC) group refers to UM-SCC-1 cells transfected with the empty vector plasmid. For the curcumin treatment groups in this set of experiments, a concentration of 20 µmol/L was used.

Article Snippet: Membranes were blocked and incubated overnight at 4°C with primary antibodies against EP300 (1:1000) (EpiZyme, China), PKM2(1:1000) (EpiZyme, China), LDHA (1:1000) (EpiZyme, China), GLUT1 (1:1000) (Affinity, USA), and β-tubulin (1:2000) (Abmart, China).

Techniques: Biomarker Discovery, Over Expression, Quantitative RT-PCR, Western Blot, Negative Control, Expressing, Comparison, Control, Transfection, Plasmid Preparation, Concentration Assay

Journal: Scientific Reports

Article Title: Curcumin inhibits glycolysis via EP300 in oral squamous cell carcinoma

doi: 10.1038/s41598-026-44496-3

Figure Lengend Snippet: EP300 positively regulates glycolysis-related enzymes in OSCC cells. ( A )Protein-protein interaction network analysis using the STRING database revealed that EP300 is closely connected with key glycolytic enzymes, including PKM2, LDHA, and GLUT1. ( B ) EP300 overexpression significantly increases mRNA expression of PKM2, GLUT1, and LDHA in UM-SCC-1 cells, as measured by qRT-PCR. ( C ) Western blot analysis showing elevated protein levels of PKM2, GLUT1, and LDHA in UM-SCC-1 cells overexpressing EP300 compared with negative control (NC). Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0. 0001.The negative control (NC) group refers to UM-SCC-1 cells transfected with the empty vector plasmid.

Article Snippet: Membranes were blocked and incubated overnight at 4°C with primary antibodies against EP300 (1:1000) (EpiZyme, China), PKM2(1:1000) (EpiZyme, China), LDHA (1:1000) (EpiZyme, China), GLUT1 (1:1000) (Affinity, USA), and β-tubulin (1:2000) (Abmart, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Control, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Curcumin inhibits glycolysis via EP300 in oral squamous cell carcinoma

doi: 10.1038/s41598-026-44496-3

Figure Lengend Snippet: Curcumin Attenuates Glycolytic Enzyme Expression via EP300.( A ) Real-time quantitative PCR analysis of PKM2, LDHA, and GLUT1 mRNA expression in UM-SCC-1 cells of the indicated groups (NC, NC+CUR, EP300-oe, EP300-oe+CUR). ( B ) Western blot analysis of PKM2, LDHA, and GLUT1 protein expression in the indicated groups. Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0. 0001.The negative control (NC) group refers to UM-SCC-1 cells transfected with the empty vector plasmid. For the curcumin treatment groups in this set of experiments, a concentration of 20 µmol/L was used.

Article Snippet: Membranes were blocked and incubated overnight at 4°C with primary antibodies against EP300 (1:1000) (EpiZyme, China), PKM2(1:1000) (EpiZyme, China), LDHA (1:1000) (EpiZyme, China), GLUT1 (1:1000) (Affinity, USA), and β-tubulin (1:2000) (Abmart, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Negative Control, Transfection, Plasmid Preparation, Concentration Assay