Fig. 3. Senescent human breast fibroblasts attenuate mammary epithelial functional differentiation. (A) Ki67 immunostaining (green) of MCF-10A cells co-cultured on Matrigel with presenescent (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 200x magnification. (B) MCF-10A alveoli formed in the presence of presenescent (Presen) or senescent (Sen) hBFs were scored for having <5 (gray bars) or >5 (black bars) Ki67-positive nuclei per alveolus. (C) Sizes of DAPI-stained MCF-10A alveoli formed on Matrigel in the presence of presenescent or senescent hBFs were determined by digital quantification of images. A minimum of 50 alveoli per condition was analyzed. Shown is the average area occupied by alveoli in presenescent (Presen) or senescent (Sen) co-cultures. Error bars indicate s.e.m. (D) Western analysis of ß-casein and cytokeratin 18 expression by Eph4 cells cultured on Matrigel plus lactogenic hormones in the presence of presenescent (Presen) or senescent (Sen) hBFs. Signals were quantified by densitometry and values from the presenescent culture were set arbitrarily at 1. Shown is the mean and standard deviation from four experiments. (E) ß-casein immunostaining (red) of EpH4 alveoli cultured in the presence of presenescent (left panels) or senescent (right panels) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 400x magnification.