Figure 2
BCAR3 antagonizes Smad activation. (a) Total cell lysates from breast cancer cells were subjected to Western blot analysis for expression of BCAR3 and p130Cas. ER-, Estrogen receptor–negative; ER+, Estrogen receptor–positive. (b) MDA-MB-231 cells were transfected with a pool of two BCAR3 small interfering RNAs (siRNAs; 25 pM each), starved overnight and stimulated with 200 pM transforming growth factor β (TGFβ) 48 hours poststarvation for the indicated time periods. Levels of phospho-Smad3, total Smad2/3, BCAR3, p130Cas and phospho-p38 were examined by Western blotting. (c) Quantification of the relative signal density of phospho-Smad3 shown in b, normalized by signal density of Smad3. The results shown are from representative experiments (n = 3). *P < 0.05 by unpaired Student’s t-test. (d) Inducible MCF-7 cells were cultured with or without 1 μg/ml doxycycline (Dox), starved overnight and stimulated with 100 pM TGFβ 96 hours posttreatment for the indicted time periods. Levels of phospho-Smad3 and BCAR3 were examined by Western blot analysis (n = 2). (e) Quantification of relative signal density of phospho-Smad3 shown in d, normalized by signal density of Smad3. Results show a representative experiment (n = 2). (f) Levels of phospho-Smad3 in the nuclear extracts from the same pool of cells shown in (c) were examined by Western blot analysis (n = 2). (g) SUM-159-PT cells were seeded onto glass coverslips, transfected with FLAG-tagged AND-34 (mouse homologue of BCAR3) and starved overnight. Cells were then treated with or without 200 pM TGFβ for 1 hour and subjected to immunofluorescence microscopy and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. In the merged images, phospho-Smad3 is shown in red, FLAG-AND-34 is shown in green and cell nuclei are shown in blue (DAPI). Yellow arrows in the middle panels point at cells that express ectopic BCAR3. (h) Phospho-Smad3 signals in transfected and nontransfected cells were quantified from ten cells on at least five original LSM780 confocal microscopic images. Error bars show standard errors of the mean. An asterisk indicates a statistical difference between the two groups compared, as determined by unpaired Student’s t-test (*P < 0.05).