Fig. 1

Co-culture of breast cancer cell line with CAF. a Growth patterns of 8 primary CAF cells. Cell proliferation assays for eight CAF cells were recorded and plotted. Y axis: average number of O.D. 595 nm of each CAF cells after crystal violet staining. Standard error of mean (SEM) bars are indicated. b Fluorescent microscopic image of co-cultured MDA-MB-231 (Red) with CAF 82T (Green). Bottom panel: merged image of red and green fluorescence. c Red fluorescence of MDA-MB-231 cells cultured with or without 82T was measured using a POLARstar Omega microplate plate reader. Student t-test was performed for assessing statistical significance. d Phase contrast images of soft agar colony formation assays for individually cultured and co-cultured MDA-MB-231 and 82T cells. e The number of colonies in each microscopic field (×10) was counted for MDA-MD-231 cells and MDA-MB-231 co-cultured with 82T cells. Y axis: average number of colonies per 10 fields. SEM bars were plotted and Student t test was used for statistical analysis. f Western blot analysis using 4G10 anti-phosphotyrosine antibody to survey the phosphotyrosine levels of individually cultured MDA-MB-231 (lane 1), 82T cells (lane 2) and co-cultured MDA-MB-231 and 82T cells (lane 4). Lane 3: individually cultured MDA-MB-231 and 82T cells were lysed and then mixed for phosphotyrosine western blot analysis. Arrows indicate the alteration of tyrosine phosphorylation induced by co-culture that are visible in the western blot