Fig 2: Notch2HIGH breast cancer cells. a Size, granularity and b percentage of MDALUC Notch2HIGH cell subpopulation after staining with anti-Notch2 antibody, analysed by flow cytometry. c Percentage of Notch2HIGH MDALUC cell subpopulation measured after magnetic-activated cell sorting (MACS). d Notch2HIGH MDALUC cells analysed in overt osteolytic breast cancer bone metastases. White arrowhead, Notch2HIGH cell. e Triple immunofluorescence in the tibias of mice injected into the medullary cavity with breast cancer 4T1 cells. f Quadruple immunofluorescence in the tibias of the mouse model of MDA-MB-231 cell dormancy and g in the mouse model of chemotherapy-induced dormancy. B bone. h Graphical scheme of MACS-sorted Notch2HIGH and Notch2LOW MDALUC injection in 5-week-old female immunocompromised CD1 nu/nu mice. i Mouse body weight measured 6 weeks after cancer cell injection. j Micro- computed tomographic analysis of bone volume over total tissue volume (BV/TV) in tibias injected with tumour cells. k Immunofluorescent human cytokeratin area over tissue area, l percentage of metastatic area over total tissue area and m number of multicellular metastases per mm2 in the histological sections of livers collected from the mice intratibially injected with tumour cells shown in i and j. Imaging and data (mean ± SD) represent the results of at least four mice/group or three independent in vitro experiments. Statistical analyses: two tails’ unpaired t test

Fig 3: Spindle-shaped N-cadherin+/CD45- osteoblasts (SNOs) inhibit breast cancer cell proliferation in vitro through the Notch pathway. a MDAGFP cells were seeded onto magnetic-activated cell-sorted (MACS) SNOs and NON-SNOs cultured for 24 h and allowed to attach for 1 h at 37 °C, followed by extensive washing. Number of MDAGFP cells at time = 0 (1 h of adhesion) and after 24–72 h of co-culture. b Number of MDAGFP clones/well after 72 h of culture. c Number of Ki-67-positive MDAGFP cells. d Fluorescence intensity of 4T1redPKH26 cells seeded onto SNO or NON-SNO monolayers after 24, 48 and 96 h of co-culture. e Number of Ki-67-positive 4T1 cells after 38 h of co-culture. f Notch1–4 expression in MDAGFP cells seeded onto SNOs and NON-SNOs, evaluated after 1 h of adhesion by confocal immunofluorescence. g Number of MDAGFP cells treated with Notch-1–4-specific small interfering RNAs (siRNAs), seeded onto SNOs and evaluated at time 0 (1 h of adhesion) and after 24–72 h, compared with SCR-siRNA-treated MDAGFP cells seeded onto SNOs and NON-SNOs. h MDAGFP cells were treated with the ?-secretase inhibitor dibenzazepine and then seeded onto SNOs. Cell number was evaluated after 1 h (time 0) and again after 24 h. The graph expresses the fold change vs time 0 and results are compared vs dimethyl sulfoxideDMSO-treated MDAGFP seeded onto SNOs and NON-SNOs. i Transcriptional expression of the indicated haematopoietic stem cell (HSC) markers in MACS sorted Notch2HIGH and Notch2LOW MDAGFP cell subpopulations. j Flow cytometry of MDA-MB-231 cells after double staining with antibodies for Notch2 and for the HSC markers, CD34, k c-kit and l Notch1. m Morphology, n number and o size of primary mammospheres formed by MACS-sorted Notch2HIGH and Notch2LOW MDALUC subpopulations. p Morphology, q number and r size of secondary mammospheres obtained after trypsinisation and re-plating of the primary mammospheres. Yellow arrowhead, mammosphere. s CD44 and CD24 expression by flow cytometry after double staining of MDALUC cells with anti-Notch2 antibody. Imaging and data (mean ± SD) represent the results of at least three independent in vitro experiments. Statistical analyses: (a, d, g) Non-linear regression fitting and F-test, (b, c, e, f, h, i, n, q) two tails’ unpaired t test (o, r) Gaussian curve regression fitting and F-test