Normalization and statistical analysis of microarray data

Raw data (CEL files) from 16 publicly available microarray series were collected from the Gene Expression Omnibus (GEO) database of National Center for Biotechnology Information (NCBI). Both Affimetrix platforms used cover an identical set of 154 genes encoding ABPs. For other genes, only probesets present in both Affimetrix platforms were analysed. R Programming Language for Statistical Computing 2.14.1 (22-12-2011) along with Bioconductor 2.10 packages was used to perform all the calculations55. Raw data were subjected to the frozen Robust Multi-Array Average (fRMA) algorithm56, followed by summarization based on multi-array model, fitted using the Median Polish algorithm. ComBat algorithm57 was used to decrease the non-biological experimental variation or batch effect for each gene independently. Expression values were transformed in log2, and probesets were mapped using the Entrez gene probeset definition58. The Pathway-Express tool from the Onto-tools (http://vortex.cs.wayne.edu) was used to assess biological processes and pathways affected at different stages of ER+ breast tumour samples. Genes encoding for human ABPs were selected from the UNIPROT database on January 2013, using the following key words: ‘actin-binding protein’; Organism: ‘9606’; localized on ‘cytoskeleton’; reviewed: ‘yes’. The list was verified based on the presence of an actin-binding domain.

Fly strains and genetics

Fly stocks used were nub-Gal4 (ref. 59), sd-Gal4 (ref. 60), Src64BUY1332 (ref. 61); UAS -p35 (ref. 62), UAS-arp3-IRKK108951, UAS-arpc5-IRKK102012, UAS-Tm2-IRKK107970, UAS-fhos-IRGD10435 (Vienna Drosophila Research Center , VDRC), UAS-ena-IRJF01155, UAS-enaS187A (ref. 63) UAS-shot-IRGL01286, UAS-GFP::arp3 (ref. 64), UAS-ena (ref. 65); UAS-shotL(A)-GFP (ref. 66), UAS-EGFP-fhosΔB (ref. 67). All crosses were maintained at 25 °C. Male and female larvae were dissected at the end of the third instar.

Cell lines, culture conditions and drug treatments

The MCF10A-ER-Src (ER-Src) and MCF10A-PBabe (PBabe) cell lines were kindly provided by K. Struhl15. The presence or absence of the ER-Src fusion construct was confirmed by PCR in the ER-Src and PBabe cells using three independent pairs of primers (5′-Src-ERB GGGAGCAGCAAGAGCAAGCCTAAG and 3′-Src-ERB CGGGGGTTTTCGGGGTTGAGC or 5′-Src-ERF GTGGCTGGCTCATTCCCTCACTACA and 3′-Src-ERF GCACCCTCTTCGCCCAGTTGA or 5′-Src-ERE AGAGGGTGCCAGGCTTTGTG and 3′-Src-ERE GGGCGTCCAGCATCTCCAG). The presence of Mycoplasma contamination was tested in both cell lines by PCR using the primers 5′-ACTCCTACGGGAGGCAGCAGT-3′ and 5′-TGCACCATCTGTCACTCTGTTAACCTC-3′, which amplifies the spacer regions between the 16S and 23S ribosomal RNA genes. None of the cell lines tested positive for Mycoplasma contamination. Cells were grown in a humidified incubator at 37 °C, under a 5% CO 2 atmosphere in DMEM/F12 growth medium (Invitrogen, 11039-047), supplemented with 5% horse serum (Invitrogen 16050-122), previously stripped of hormones through dextran-coated charcoal incubation (Sigma C6241), 20 ng per ml of EGF (Peprotech, AF-100-15), 10 μg per ml of insulin (Sigma, I9278), 0.5 μg per ml of hydrocortisone (Sigma, H-0888), 100 ng per ml of cholera toxin (Sigma, C-8052), and penicillin/streptomycin (Invitrogen, 15070-063). To treat cells with 4OH-TAM or EtOH, 50% confluent cells were plated and allowed to adhere for at least 24 h before treatment with 1 μM 4OH-TAM (Sigma, H7904) or with identical volume of EtOH for the time periods indicated in the text. For YAP/TAZ localization assessment, cells were cultured in plain DMEM/F12 during the whole course of the experiments. Plain DMEM/F12 cell culture medium, containing EtOH alone or 4OH-TAM diluted in EtOH, was replaced 4, 8 and 12 h after the beginning of the experiments. To assess ERK activation and Cyclin D1 expression, cells were serum-starved for 16 h in plain DMEM/F12, before treatment with DMSO or 2.5 μM of the MEK inhibitor PD184352 (Sigma, PZ0181) or 10 μM of Blebbistatin (Sigma, B0560), or without any additive for 1 h. Culture medium was then replaced with plain DMEM/F12, containing EtOH or 4OH-TAM, and DMSO or 2.5 μM of PD184352 or 10 μM of Blebbistatin or without any additive, before analysis 12 h later.

3D matrigel cultures

Lab-Tek II plates (Lab-Tek II, #155409) were coated with 20 μl Matrigel (BD Biosciences, 356231). A total of 1,000 cells were suspended in 100 μl Matrigel and overlaid in wells. After polymerization at 37 °C, 500 μl of cell culture media was added and replaced every 3 days. Cells were grown for 14 days and treated with cell culture medium containing serum, growth factors and EtOH or 4OH-TAM. Acini were imaged by transmitted light and DIC optics for maximum contrast using a Leica DMI6000 inverted microscope coupled to a Hamamatsu Orca Flash 4.0 sCMOS camera with a Leica 10 × 0.3 numerical aperture PLAN FLUOR dry objective. Image processing and analysis were performed using Fiji (ImageJ package distribution). For size quantification, acini edges were outlined using the Find Edges function, converted to a binary mask before measurement using the area function.

Time-lapse imaging

For time-lapse imaging, samples were maintained at 37 °C in a controlled unit with 95% relative humidity, 5% CO 2 . Transmitted light DIC images were acquired on a Yokogawa CSU-X Spinning Disk confocal scan head, coupled to a Nikon Ti microscope, using a 10 × 0.3 numerical aperture objective, with an Andor iXon+ EMCCD camera at 30 or 15 min (min) intervals for a period of 60 h for 3D Matrigel culture or 2D, respectively. Images were acquired using MicroManager 1.4.15 acquisition software. Processing and compression were performed using Fiji. To quantify cell velocity, cell tracking was performed using the Manual Tracking plugin function available in the Fiji software package (quote: http://imagej.net/Citing). Total track length of cell movement was measured and used to quantify the average velocity of each cell per condition.

Immunofluoresence analysis and quantifications

For wing imaginal discs, staining was performed by dissecting larvae in phosphate buffer at pH 7 (0.1 M NA 2 HPO 4 and 0.1 M NAH 2 PO 4 at a 72:28 ratio). Discs were then fixed in 4% formaldehyde in PEM (0.1 M PIPES (pH 7.0), 2 mM MgSO 4 , 1 mM EGTA) for 15–30 min, rinsed in phosphate buffer 0.2% Triton for 15 min and incubated overnight with mouse anti-Ena (1:50, 5G2, Developmental Studies Hybridoma Bank; 1:50) at 4 °C. Discs were then rinsed three times for 10 min in phosphate buffer 0.2% triton, incubated for 1 h with TRITC Donkey anti-mouse (Jackson ImmunoResearch, 715-025-150) in phosphate buffer 0.2% triton supplemented with 10% horse serum and rinsed three more times for 10 min before being mounted in Vectashield (Vector Labs, H-1000). For Phalloidin staining, discs were dissected in phosphate buffer at pH 7 (0.1 M NA 2 HPO 4 and 0.1 M NAH 2 PO 4 at a 72:28 ratio). Discs were then fixed in 4% formaldehyde in PEM (0.1 M PIPES (pH 7.0), 2 mM MgSO 4 , 1 mM EGTA) for 15–30 min, rinsed in phosphate buffer 0.2% Triton for 15 min and incubated for 1 h at room temperature with Rhodamine-conjugated Phalloidin (Sigma, P-1951) at 0.3 mM in phosphate buffer 0.2% triton X-100 supplemented with 10% horse serum before being rinsed and mounted in Vectashield (Vector Labs, H-1000). Fluorescence images were obtained with an LSM 510 Zeiss or Leica SP5 live confocal microscope using a × 10 or × 20 dry objective. The NIH Image J program was used to quantify wing disc area. Each disc was outlined and measured using the Area function, which evaluates size in square pixels. To quantify the ratio of the nub>GFP domain over the total wing disc area, the ratio between the area of the GFP domain and the area of the whole disc domain, measured using the Area function for each disc, was calculated.

MCF10A cells were plated in poly-L-lysine-coated coverslips (Sigma, P-8920). To stain cells with the anti-β-CYA and anti-γ-CYA, epithelial cell monolayers were fixed with prewarmed 1% paraformaldehyde in DMEM for 30 min, followed by 5 min permeabilization with methanol at −20 °C. Cells were then rinsed slowly with sequential methanol dilution in phosphate-buffered solution (PBS). For all other fluorescence staining, cells were fixed in 4% paraformaldehyde in PBS at pH 7 for 10 min and permeabilized with TBS-T (TBS—0.1% Triton X-100) at room temperature. For all staining, cells were blocked in TBS-T supplemented with 10% BSA for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in blocking solution. Coverslips were then washed three times with TBS and incubated with secondary antibodies and with Rhodamine-conjugated Phalloidin (Sigma, P-1951) at 0.3 mM in blocking solution for 1 h at room temperature. After three washes in TBS, cells were stained with 2 μg ml−1 DAPI (Sigma, D9542) for 5 min in TBS, washed again with TBS and mounted on Vectashield. The following primary antibodies were used: anti-EVL (1:50; Sigma, HPA018849), anti-activated Src (1:100; Invitrogen, 44-660G), anti-phospho-Myosin Light Chain 2 (Thr18/Ser19) (1:200; Cell Signaling, 3674), anti-Paxillin (1:200; BD Pharmingen, 610051), anti-β-CYA (1:50; mAb 4C2, IgG1, a gift from C. Chaponnier24), anti-γ-CYA (1:100; mAb 2A3, IgG2b, a gift from C. Chaponnier24) and anti-YAP (1:200; Santa Cruz Biotechnology, sc101199). The secondary antibodies used to detect anti-β-CYA and anti-γ-CYA were anti-mouse IgG1 FITC-conjugated (1:50; Invitrogen, A21240) and anti-mouse IgG2b alexa 647-conjugated (1:50; Invitrogen, A21141), respectively. The secondary antibodies used to detect all other primary antibodies were IgG TRITC or FITC or Alexa Fluor 647-conjugated (1:200, Jackson Immunoresearch). Fluorescence images were obtained on a Leica SP5 confocal coupled to a Leica DMI6000, using the 63 × 1.4 HCX PL APO CS Oil immersion objective. Quantifications of stress fibres anisotrophy were performed using Fibril Tool plugin for NIH Image J program, where each individual cell was outlined and considered as the region of interest. For quantification of cell fraction with high basal F-actin levels, images were processed and analysed using Fiji. For each cell, the DAPI channel was used to outline the nucleus area. Quantification of F-actin intensity signal was performed for each cell within the nucleus area on projected images of the basal cell surface. The F-actin signal intensity of each cell was then semi-quantitatively scored. Cells were considered negative if their F-actin signal intensity was lower than the average of the five lowest F-actin intensity values. Cells were considered positive if their F-actin signal intensity was higher than three times the average of the five lowest F-actin intensity values. For quantification of YAP/TAZ localization, cytoplasmic localization was defined, as YAP/TAZ was completely absent in the nucleus.

shRNA adenovirus infection and number of cells

ER-Src cells were plated in six-well plates to reach 30% confluence by the time of infection. shEVL or shScr virus-containing media were added at a multiplicity of infection of 103 plaque-forming unit per cell, reaching 100% of efficiency of gene delivery. Media were changed after 24 h. Treatments with cell culture media containing EtOH or 4OH-TAM were performed 72 h after infection.

EVLshRNA#1: 5′-GCCAAATGGAAGATCCTAGTACTCGAGTACTAGGATCTTCCATTTGGC-3′;

EVLshRNA#2: 5′-ACGATGACACCAGTAAGAAATCTCGAGATTTCTTACTGGTGTCATCG-3′;

ShScr: 5′-GACACGCGACTTGTACCACTTCAAGAGAGTGGTACAAGTCGCGTGTCTTTTTTACGCGT-3′.

The number of cells for each experimental condition was quantified 36 h after treatment using the Scepter 2.0 Handheld Automated Cell Counter.

Soft agar colony assay

Cells (5 × 103) in cell culture media containing no EGF were mixed with 0.36% gelling agarose (Sigma, A9045) and plated on top of a solidified layer of 0.7% agarose in cell culture media with no EGF. Cells were fed every 6–7 days with cell culture media with no EGF. The number of colonies was counted 15–21 days later.

Immunoblotting analysis and quantification

Protein extracts from MCF10A cells were obtained by scraping cells in TRIS lysis buffer, containing protease (Roche, cOmplete Tablets, 4693159001) and phosphatase (Roche, PhosSTOP Tablets, 4906837001) inhibitors, and lysed for 20 min on ice. Protein extracts from wing imaginal discs were obtained by dissecting discs in phosphate buffer at pH 7 (0.1 M NA 2 HPO 4 and 0.1 M NAH 2 PO 4 at a 72:28 ratio). Discs were then lysed in 10 μl 2% SDS. Sample Buffer 2 × was then added to cells or wing imaginal disc lysates before boiling the mixture for 5 min and clearing by centrifugation at 16,168 g at 4 °C for 30 min. Protein was resolved by SDS–PAGE electrophoresis and transferred to PVDF membranes (Amersham Pharmacia, 10600023). Membranes were blocked with 5% milk in TBS 0.1% Tween 20 and incubated with the following: rabbit anti-activated Src (1:1,000; Invitrogen, 44-660G), rabbit anti-EVL (1:250; Sigma, HPA018849), rabbit anti-GAPDH (1:2,000; Santa Cruz, 2D4A7), mouse anti-phospho-p44/42 MAPK (1:2,000; Cell Signaling, 9106), rabbit anti-p44/42 MAPK (1:1,000; Cell Signaling, 9102), rabbit anti-phospho-Myosin Light Chain 2 (Ser19) (1:500; Cell Signaling, 3671), rabbit anti-Actin (1:500; Sigma, A2066), mouse anti-Ena (1:200; Developmental Studies Hybridoma Bank, 5G2) and rabbit anti-α-Tubulin (1:1,000; Sigma, T6199). The specificity of the anti-Ena antibody has been verified on wing disc extracts carrying UAS-enaS187A (ref. 51) and nub-Gal4. Detection was performed by using HRP-conjugated antisera (Jackson Immunoresearch) and Enhanced Chemi-Luminescence (ECL) detection (Thermo Scientific, 32106). Western blots were quantified using the Image Studio Lite program. Uncropped scans of the most relevant western blots can be found in Supplementary Fig. 7.

Real-time PCR analysis

One microgram of purified RNA samples was reverse-transcribed using intron–exon-specific primers. Real-time qPCR were performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences) in 384-well plates in the Bio-Rad, #1725125. The relative amount of EVL, Cyclin D1, CTGF or ANKRD1 mRNA was calculated after normalization to the GAPDH transcript. Primers used for EVL were 5′-GAAGAGTCCAACGGCCAGAA-3′ and 5′-ACTGGGAGGCTGCTTTTCTC-3′. Primers used for GAPDH were 5′-CTCTGCTCCTCCTGTTCGAC-3′ and 5′-ACCAAATCCGTTGACTCCGAC-3′. Primers used for CTGF were 5′-CTCGCGGCTTACCGACTG-3′ and 5′-GGCTCTGCTTCTCTAGCCTG-3′. Primers used for ANKRD1 were 5′-TAGCGCCCGAGATAAGTTGC-3′ and 5′-GGTTCAGTCTCACCGCATCA-3′. Primers used for Cyclin D1 were 5′-GATCAAGTGTGACCCGGACTG-3′ and 5′-CCTTGGGGTCCATGTTCTGC-3′.

Cell cycle profile

Sub-confluent cells were cultured overnight in media without EGF. Cells were then incubated in culture media containing EGF and EtOH or 4OH-TAM for 12 h, fixed with 70% EtOH and stained with 10 μg ml−1 of propidium iodide (Sigma, P4170). Cell cycle profiles were obtained using FACS Calibur. Flow Logic software was used for quantification of the percentage of S-phase cells.

G/F actin assay

The G-actin/F-actin In vivo Assay Kit (Cytoskeleton, Denver, CO, USA, BK037) was used to quantify the F- and G-actin pools. Cells were harvested by scraping in lysis buffer. Cell lysates were then incubated for 10 min at 37 °C and centrifuged at 350 g at room temperature for 5 min to pellet unbroken cells. Supernatants were then centrifuged at 90,000 g for 90 min at room temperature to pellet F-actin and leave G-actin in the supernatant. Supernatants were then removed and kept on the side in 5 × SDS sample buffer, while pellets were incubated in F-actin depolymerization buffer on ice for 1 h to allow actin depolymerization to occur before the addition of 5 × SDS sample buffer. G- and F-actin fractions were then analysed by immunoblotting analysis and quantified using the Image Studio Lite program, as the ratio between F- and G-actin levels for each experimental condition.

Atomic force microscopy

For the AFM indentation experiments a Nanowizard I (JPK Instruments, Berlin) equipped with a CellHesion module was used. Arrow-T1 cantilevers (Nanoworld, Neuchatel, Switzerland) were modified with polystyrene beads (radius 2.5 μm, Microparticles GmbH, Berlin, Germany) with the aid of epoxy glue to obtain a well-defined spherical indenter geometry and decrease local strain during indentation. The cantilever was lowered with a speed of 5 μm s−1 until a relative set point of 2.5 nN was reached. The resulting force distance curves were analysed using JPK image processing software (JPK instruments). Force distance data were corrected for the tip sample separation and fitted with a Hertz model for a spherical indenter to extract the apparent Young’s Modulus68. A Poisson ratio of 0.5 was assumed. A 2-h time window was required for measurement of each experimental condition.

Real-time deformability cytometry

A microfluidic chip was made out of PDMS using soft-lithography and sealed with a glass coverslip after plasma surface activation. The microfluidic chip consisted of two reservoirs connected by a 300-μm-long constriction channel with a 30 μm by 30 μm cross-section. A row of filter posts at the inlet prevents the channel from clogging by cell clumps and debris. For measurement, cells were re-suspended in a 0.5% methylcellulose solution in PBS at a concentration of 4–5 × 106 cells per ml. The cell suspension was drawn into 1 ml syringes and connected to the chip by polymer tubing. Cells were pumped with a syringe pump at a constant flow rate of 0.16 μl s−1 (0.04 μl s−1 sample+0.12 μl s−1 sheath) for 2 min before collecting data, to stabilize flow. Data (typically, a minimum of 2,000 events) were acquired in real time with a high-speed CMOS camera (MC1362; Mikrotron, Unterschleissheim, Germany), operating at 2,000 f.p.s., and illuminated by a high-power LED (CBT-120, 462 nm; Luminus devices, Woburn, MA, USA) through a × 40 objective of an inverted microscope (Axiovert 200M, Carl Zeiss, Oberkochen, Germany) at the end of the 300-μm-long constriction channel where the cell shape had reached steady state. An image-processing algorithm implemented on C++LabVIEW was used to determine the cell cross-sectional area and circularity, defined as , where A is the projected area of the cells and l its perimeter. An analytical model69 was used to calculate the hydrodynamic flow profile around a moving cell in confinement, couple the resulting surface stress to a linear elastic model of a homogeneous isotropic sphere, and extract the elastic modulus for comparison with the observed cell parameters, assuming a constant viscosity of the surrounding fluid of 15 mPa.

Breast carcinoma series

Formalin-fixed and paraffin-embedded breast tumour samples were obtained from a Pathology Laboratory of Araçatuba (Veronese Patologia e Citologia Araçatuba, Brazil), under patient informed consent and with ethical approval by the lab research review boards, implying that the spare biological material, which has not been used for diagnosis, could be used for research. This is in accordance with the national regulative law for the handling of biological specimens from tumour banks, being the samples exclusively available for research purposes in retrospective studies, as well as under the International Helsinki Declaration. All breast tumour cases were previously characterized with regard to ER, progesterone receptor (PR), epidermal growth factor receptor (EGFR), HER2, CK5, P-cad and the antigen Ki-67, in addition to patients’ age and clinicopathological features, such as grade, tumour infiltrating lymphocytes (inflammation) and lymph nodes status. Breast tumour components were classified into luminal A and B, HER2-overexpressing, and basal-like carcinomas, according to the immunoprofile70. Twenty-four cases of normal breast samples derived from reduction mammoplasties or adjacent to tumour tissue were included in this study. All morphological and immunohistochemical assessments were conducted by a pathologist (AP). The study was conducted under the national regulative law for the handling of biological specimens from tumour banks, the samples being exclusively available for research purposes in retrospective studies.

Immunohistochemistry and quantification

IHC was performed using the Ultravision Detection System Anti-polyvalent HRP (Lab Vision Corporation) for EVL. Antigen unmasking was performed using a commercially available solution of citrate buffer, pH 6.0 (Vector Laboratories), at a dilution of 1:100 at 98 °C for 30 min. After the antigen retrieval procedure, slides were washed in PBS and submitted to blockage of the endogenous peroxidase activity by incubation in methanol (Sigma-Aldrich) containing 3% hydrogen peroxide (Panreac). Slides were further incubated with the primary antibody for EVL (1:100; Sigma, HPA018849). The IHC reaction was revealed with diaminobenzidine (DAB) chromogen (DakoCytomation). The positive control was a tonsil sample, which was included in each run, to guarantee the reliability of the assays. Non-neoplastic breast tissues, as well as normal breast surrounding the neoplastic cells, were considered internal controls. EVL expression was mainly found in the cell cytoplasm. The normal breast tissue and each component of breast carcinoma were semi-quantitatively scored with regard to the intensity (I) of staining as negative (0, no staining), weak (1, diffuse weak), moderate (2, moderate staining) or strong (3, defined as strong staining), and the extent (E) of stained cells was evaluated as a percentage. For each case, a staining score was obtained by multiplying I x E (range 0-300). Cases with a staining score ≤100 were considered negative and cases with a score >100 were classified as positive. The TMA cores measured 2 mm in diameter and included a representative area of the tumour; up to three cores of each case were represented in the TMA. In the cases where in situ and invasive carcinomas were present in the same core (n=71), the samples were classified using the nomenclature increase/decrease/equal EVL expression according to the change in IxE score.

Sample size

No statistical methods were used to predetermine the sample size. Microarray data of 903 neoplastic breast lesions, including 12 ER+ ADH, 22 ER+ DCIS, 68 ER+ IDC1, 189 ER+ IDC2, 273 ER+ IDC3, 44 ER− IDC2 and 299 ER− IDC3, were compared to microarray data of 255 normal breast tissues. For Drosophila wing disc growth measurements, 101 samples from four independent nub-Gal4, UAS-GFP>UAS-Src64BUY1332, UAS-p35 crosses (biological replicates) were analysed. For all other crosses, 17–96 samples from two independent crosses (biological replicates) were analysed. Three biological replicates were used for quantification by western blots of pERK, total ERK, pMLC, ER-pSrc, EVL, Ena or actin levels. Six biological replicates were used for quantification of ER-pSrc in ER-Src cells treated with EtOH or TAM for 4, 12, 24 or 36 h. Quantification of EVL protein levels in ER-Src cells knocked down for EVL was carried out from one biological replicate. Protein extracts from wing imaginal discs were obtained by dissecting four discs per genetic background from two independent crosses (biological replicates). To quantify actin levels or F-/G-actin ratio, three biological replicates were used in triplicate (technical replicates). Quantifications of Cyclin D1, EVL, CTGF or ANKRD1 mRNA levels were from three technical replicates, repeated three times (biological replicates). Three biological replicates were used to quantify cell number or number of cells in S-phase of the cell cycle. Quantifications of acini size were from two biological replicates. Quantifications of number of colonies in soft agar were from three biological replicates in triplicate (technical replicates). For quantification of fibre anisotropy, between 90 and 103 cells from three biological replicates were analysed per experimental condition. To quantify the fraction of cells with high basal F-actin levels, 140 to 220 cells from two biological replicates were used. To quantify cell velocity, 100–200 cells from three biological replicates were tracked per condition. To quantify YAP/TAZ localization, eight fields per experimental conditions from two biological replicates were used. A total of 160 (2D) or 100 cells (3D) per experimental conditions from three (2D) or two (3D) biological replicates were used to measure stiffness by AFM. Two biological replicates per condition were used to measure stiffness by RT-DC. The archive of breast tumour samples includes 150 invasive breast carcinomas and 91 in situ carcinomas, from which areas of in situ and invasive carcinoma can be found in the same block for 71 patients. In addition, 24 normal breast samples were used for comparison.

All experiments were considered for quantification, with the exception of two. For quantification of ER-pSrc levels during the 36 h of cellular transformation, blots with a high degree of variance in GAPDH levels between samples or in ER-pSrc levels between biological replicates were excluded. For quantification of pMLC between ER-Src cells treated with EtOH or TAM for 12 h, three biological replicates, which did not show an increase in ER-pSrc levels and endogenous pSrc, were excluded. Blinding was used to quantify YAP/TAZ localization. No other quantification used blinding. Randomizations were used to quantify fibre anisotropy and the fraction of cells with high basal F-actin levels by selecting each field using the DAPI channel exclusively.

Statistics

Statistical analyses of EVL expression in normal, DCIS and IDC samples were conducted using IBM SPSS Statistics (Version 22.0. Armonk, NY: IBM Corp). The associations between categorical variables were tested for statistical significance using the chi-square test to compare multiple conditions or using Fisher’s exact tests to compare two conditions. Both tests evaluate the strength of the association between two categorical variables. A two-tailed significance level of 5% was considered statistically significant (P<0.05). Other statistical analyses were performed using Prism 6.0 software (GraphPad Inc.). For statistical comparison of two independent groups (Figs 1e,f, 2i and 4b,c,g; and Supplementary Figs 2C and 5) the unpaired t-test were used. For statistical comparison between more than two groups (Figs 1a,f,g, 2b–d, 5b–h, 6e,f and 7b–e; Supplementary Figs 2B,3,6B,C,E,F), one-way ANOVA tests were used. For statistical comparison of the percentage of S-phase cells between EtOH- and TAM-treated ER-Src cells over time (Fig. 1d and Supplementary Fig. 2A), two-way ANOVA tests were used. To detect differences in EVL protein levels across multiple test attempts between EtOH- and TAM-treated ER-Src cells during the 36 h of cellular transformation, a Friedman test was used. For statistical comparison of gene expression profiles between normal breast samples and breast tumour samples of different stages, grades and ER status, the LIMMA (Linear Models for Microarray Data) algorithm was used to calculate fold change (Log2R), B-statistics (lods), t-statistics and p-values. Genes selected as differentially expressed were those with a lods value higher than 0: that is, the probability of being differentially expressed was higher that 50% (ref. 71). Statistical significance of apparent Young’s moduli between EtOH- and TAM-treated ER-Src cells for each time point (Fig. 2f,g) was calculated using a simple Mann–Whitney test or the Kruskal–Wallis test with multiple comparisons for ER-Src cells expressing shScr or shEVL and treated with EtOH or TAM for 12 h. For RT-DC, statistical analysis was performed using linear mixed models. This approach allows the comparison of different treatment states, considering the reproducibility of the effect. We defined a model that allows an individual mean for each biological replicate (random intercept). In addition, the difference between the compared treatment states (ETOH and TAM) was allowed to be different for each replicate (‘random slope’). This model was then compared to a so-called ‘Null model’, which assumes that there is no difference between the treatment states. A likelihood ratio test and Wilks theorem were used to quantify the similarity of the two models, using a p value. The P-value indicates whether the null hypothesis that both models are identical is true.

Data availability

Microarray data of genes deregulated in TAM-induced ER-Src cells18 have been deposited at GEO under accession number GSE17941. Affimetrix platforms used in this study were HG-U133A and HG-U133A PLUS 2.0.; series accession numbers were GSE15852, GSE16873, GSE7390, GSE20194, GSE10810, GSE21422, GSE22544, GSE23593, GSE5460, GSE5764, GSE10780, GSE2109, GSE3744, GSE17907, GSE19615 and GSE23177. See also Supplementary Table 1. All other remaining data are available within the Article and Supplementary Files, or available from the authors upon request.