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. 2012 Aug 22;7(8):e43459. doi: 10.1371/journal.pone.0043459

Figure 4. Receptor-independent phosphorylation of Smad3 is sensitive to an increase in the volume of mitotic cells and occurs upon GFP-Smad3 over-expression.

Figure 4

A, α-pSmad3C, α-tSmad3 and α-clathrin immunoblot of ES-2 cells treated with 2ME2 or vehicle, hypotonic medium (2 h; as described in Materials and Methods) or their combination. 2ME2-arrested cells treated with hypotonic medium show 0.31±0.09 fold lower levels of the (pSmad3C/tSmad3)/clathrin ratio (n = 4; p<0.05, 1-tailed t-test), and 1.94±0.23 fold increase in tSmad3/clathrin ratio (n = 4; p<0.01, 1-tailed t-test), as compared to 2ME2-arrested cells grown in isotonic medium. B, Confocal micrographs of tubulin and DNA of 2ME2-arrested ES-2 cells, treated or not with hypotonic medium. Bar, 10 µm. Numbers represent the percentage of cellular tubulin that localizes to the spindle. In arrested cells treated with hypotonic medium, this percentage was 0.44±0.09 fold lower as compared to arrested cells grown in normal medium (n = 6; p<0.03, 1-tailed t-test). C, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, transfected with GFP-Smad3 or GFP constructs and treated with SB431542 (6 h) or vehicle. D, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, transfected with GFP-Smad3 and treated with reversine (5 µM, 2 h) or vehicle. Transfected cells treated with reversine showed 0.58±0.06 fold lower levels of (pGFP-Smad3C/tGFP-Smad3)/clathrin ratio (n = 4; p<0.05, 1-tailed t-test), as compared to untreated cells.