Figure 4
Characterization of immortalized mammary epithelial cell cultures with different Smad genotypes. (a) Western blot analysis of Smad2 and Smad3 expression in different immortalized mammary epithelial cell (IMEC) cultures. Smad2 was excised from IMECs that were homozygous for the conditional allele of Smad2 (Smad2fl/fl) by ex vivo exposure to adenovirus expressing Cre recombinase. Adenovirus expressing LacZ was used as a control for nonspecific effects of viral transduction. Mouse embryo fibroblasts (MEFs) derived from germline Smad2 knockout mice (S2KO) were used as a positive control for complete Smad2 deletion. β-actin used as a loading control. WT, wildtype. (b) Morphology of Smad null cultures under nonpermissive conditions. White scale bar = 100 μm. (c) Long-term growth curves for the various IMEC cultures grown under permissive conditions. Growth plotted as cumulative population doublings. Results are representative of two independent experiments. (d) Short-term growth curves for the various IMEC cultures after shifting to nonpermissive conditions. Results are the mean ± standard error of the mean for three independent experiments. (e) Western blot of Smad phosphorylation in response to transforming growth factor beta (TGFβ) (2 ng/ml for 30 minutes) in the IMECs of different genotypes. Note that the phospho-Smad3 antibody also recognizes phospho-Smad1, phospho-Smad5 and phospho-Smad8, which comprise the upper of the two bands on the phospho-Smad3 blot. (f) Quantitative RT-PCR assessment of TGFβ receptor type II (TGFBR2) mRNA. Data are normalized to the wildtype genotype. *P < 0.05 for expression difference between specified genotype and wildtype control (Student's t test). AU, arbitrary units.