Fig 2: 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

Fig 3: 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

Fig 4: 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 lg2 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

Fig 5: 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. 5c 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