Wnt signalling in normal breast cell lines (N), ER-ve (T−) and ER+ve (T+) breast cancer cell lines.

<p>A) mRNA expression was normalised to house keeper genes. Cluster analysis was performed and data displayed in a heatmap: decreased (green) or increased (red) expression compared to the mean mRNA expression. (Red) ↑↑ Indicates significant increased expression (<0.05) (red) ↑ indicates a trend towards increased expression. (Green) Indicates significant increased expression (<0.05) (green) indicated a trend towards increased expression. B) Protein expression of Lef 1 and B-actin (housekeeper) in normal breast cell lines (N), ER-ve (T−) and ER+ve (T+) breast cancer cell lines.</p

Analysis of mRNA expression in primary human breast cancer samples.

<p>Expression data was combined from multiple studies into a combined cohort of ERBB2 (n = 194) Basal (172) Luminal A (n = 336) and Luminal B (161) and normal-like (n = 244) subtypes of breast cancer. A) mRNA expression data was displayed in a heatmap: decreased (green) or increased (red) expression compared to the mean mRNA expression. % of samples with high gene expression are detailed and denoted as green when expression is decreased and red when expression is increased. B) Kaplan Meier plots Wnt gene expression show the years of recurrence free survival, where red indicates lower gene expression and blue indicates higher gene expression. * Red ≤upper quartile: Blue >upper quartile ** Red ≤lower quartile: Blue >lower quartile. P values were generated and significant results were highlighted in red.</p

Modulation of Wnt signalling in normal and primary breast cancer samples (Normal n = 3; ER+ve n = 3; ER-ve n = 3).

<p>Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) primary normal breast cells (Wnt3a) B) ER+ve primary breast cancer cells (Wnt3a) C) ER-ve primary breast cancer cells (Wnt3a) D) primary normal breast cells (DKK1) E) ER+ve primary breast cancer cells (DKK1) F) ER−ve primary breast cancer cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * >0.01 ** >0.001 generated by a T-test. G) Image of a normal primary mammosphere H) Image of an ER positive primary tumour mammosphere I) Image of an ER negative primary tumour mammosphere. Scale bar represents 50 µM.</p

Gene expression analysis of Wnt signalling in monolayer (Mono) and anoikis resistant (AR) cells of normal breast cell lines (N), ER-ve and ER+ve breast cancer cell lines.

<p>A) Protein expression of activated B-catenin (unphosphorylated), Lef1, Axin2, DKK1 and B-actin (housekeeper) in MCF7 monolayer and AR cells. B) Cluster analysis was performed using the fold change in expression between Mono and AR cells. Data is displayed in a heatmap represented by either decreased (green) or increased (red) expression compared to the mean mRNA expression. (Red) ↑↑ Indicates significant increased expression (<0.05) (red) ↑ indicates a trend towards increased expression. (Green) Indicates significant increased expression (<0.05) (green) indicated a trend towards increased expression.</p

Modulation of Wnt signalling in normal and breast cancer cell lines.

<p>Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) MCF10a cells (Wnt3a) B) MCF7 cells (Wnt3a) C) MDA-MB-231 cells (Wnt3a) D) MCF10a cells (DKK1) E) MCF7 cells (DKK1) F) MDA-MB-231 cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * <0.01 ** <0.001 generated by a T-test. G) Image of a MCF10a mammosphere H) Image of an MCF7 mammosphere I) Image of an MDA-MB-231 mammosphere. Scale bar represents 50 µM.</p

Additional file 2: Figure S1. of Intermittent energy restriction induces changes in breast gene expression and systemic metabolism

Changes in gene expression due to CER are also changed in approximately half of the IER participants, but are not clearly correlated with lymphocytes. The heatmap colours show log2 fold change in gene expression of the baseline samples relative to the post-diet samples (green = downregulated, red = upregulated, black = no change). The arrows show subjects predicted to have an energy restriction (orange = CER, blue = IER) or unchanged profiles (grey). Similar results were obtained when considering all probe sets on the array. (XLSX 268 kb

Additional file 1: Table S1. of Intermittent energy restriction induces changes in breast gene expression and systemic metabolism

Dietary intake at baseline and after four to five weeks of IER, during restricted and unrestricted days of IER (n = 23). Table S2. a LCMS+ changes in identified serum metabolites after four to five weeks of IER, on restricted and unrestricted days of IER. b LCMS– changes in identified serum metabolites after four to five weeks of IER on restricted and unrestricted days of IER. c GCMS changes in identified serum metabolites after four to five weeks of IER on restricted and unrestricted days of IER. d GC-MS changes in identified urine metabolites after four to five weeks of IER on restricted and unrestricted days of IER. Table S3 Comparison of baseline characteristics, physical and metabolic changes between the molecular responders and non-responders identified by breast tissue gene expression following IER. Table S4 Percentage change in body weight, BMI, adiposity, lipid and hormone levels with four to five weeks of IER in comparison with four to five weeks of 60 % continuous energy restriction. (PPTX 233 kb