Figure 6
Functional effects of exposure to BPA and B(a)P on the response of immature cell to BMP2 and BMP4. (a) Bar graph showing cell proliferation of MFC10A cells exposed 60 days to BPA or B(a)P (10−10 M) and treated for 4 days with BMP2 or BMP4 (15 ng/ml). Data are represented as mean +/− S.E.M., n=21. (b) Column scatter plot showing the number of spheres obtained after 1 week per 1000 seeded cells. One hundred single MFC10A cells exposed 60 days to BPA or B(a)P (10−10 M) and treated for 4 days with BMP2 or BMP4 (15 ng/ml) were seeded onto 96 ultra-low attachment plates (150 μl medium/well), each value represents the mean of one experiment (mean of 30 wells). Data are represented as mean +/− S.E.M., n=4. (c) After 60 days of exposure to BPA or B(a)P (10−10 M) and 4 days of treatment with BMP2 or BMP4 (15 ng/ml), 200 cells per well were seeded onto 12-well plates in triplicate to perform E-CFC assays for 5–6 days. Bar graph showing the number of different types of E-CFCs obtained per 10 000 seeded cells (mixed colonies, mixed-CFC; myoepithelial colonies, myo-CFC; luminal colonies, lu-CFC). Data are represented as mean +/− S.E.M., n=6. (d,e) Cells were exposed 60 days to BPA (10−10 M) (d) or to B(a)P (10−10 M) (e) and treated for 4 days with BMP2 (15 ng/ml), before conducting qPCR analyses to detect the level of expression of differentiation markers. Column scatter plot showing arbitrary units of the ratio to control cells unexposed to pollutants (CT) but treated for 4 days with BMP2 (15 ng/ml). Data represent mean +/− S.E.M., n=5. (f,g) Cells were to BPA (10−10 M) (f) or to B(a)P (10−10 M) (g) and treated for 4 days with BMP4 (15 ng/ml), before conducting qPCR analyses to detect the level of expression of differentiation markers. Column scatter plot showing arbitrary units of the ratio to cells unexposed to pollutants (CT) treated for 4 days with BMP2 (15 ng/ml). Data represent mean +/− S.E.M., n=5. (h) Bright-field (left) or immunohistochemistry (right) images at day 10 of 3D structures generated in the TDLU assay from unexposed control cells (CT), BPA or B(a)P previously exposed (60 days, (10−10 M)) cells treated for 4 days with BMP2 or BMP4 (15 ng/ml). Data are representative of n=9 independent experiments, scale bars=100 μm
