Fig. 4.

Dorsomorphin-mediated inhibition of BMP signaling reduces proliferation and alters actin cytoskeleton organization of ES-2 cells. a ES-2 cells, treated or not with 8 µM dorsomorphin (48 h), were serum-starved (2 h) and stimulated (or not) with 100 ng/ml BMP2 (45 min). Cell lysates were separated by SDS-PAGE and immunoblotted for p-Smad1/5/8, t-Smad1/5/8, and clathrin (loading control). b Dorsomorphin inhibits proliferation of ES-2 cells. 300,000 ES-2 cells were plated in 6-well plates and treated with indicated concentrations of dorsomorphin. Graph depicts average ± s.e.m. of number of cells at 48 h in each condition (n = 3; dashed line depicts initial cell number). c, d Dorsomorphin alters organization of actin cytoskeleton in ES-2 cells. ES-2 cells, treated (or not) with 4 µM dorsomorphin (48 h), were stained with Alexa-546-conjugated phalloidin and DAPI. c Representative micrographs of cell fields. Bar 100 µm. d Quantification of the distribution of intensity of actin signals. Alexa-546 Phalloidin signals were intensity-based segmented into three intensity categories. Graph depicts the percentage of Alexa-546 Phalloidin signal in each category from multiple fields (n = 12). Increase in low intensity category, and concomitant decreases in higher intensity categories were significant for dorsomorphin vs. untreated cells (p < 0.05; n = 12)