Fluorescence microscopy in human monolayer MSC. Cells were incubated with 10 ng/ml TGF-β3 and/or 10 μM ROCK inhibitor Y-27632 and analysed at days 1 and 3. Representative images show phalloidin staining of the actin cytoskeleton, which is disrupted upon ROCK inhibition, nuclear staining with DAPI, and Smad 2/3 immunostaining with nuclear translocation upon TGF-β3 and TGF-β3 plus Y-27632 stimulation. Differences between groups are more clearly visible on day 1. The graphs represent the data from quantitative image analyses in cells from 3 donors, indicating a relatively constant amount of total Smad 2/3 (left) and nuclear translocation upon incubation with TGF-β3 and Y-27632 (right). Bars represent the median values and error bars the 95% confidence intervals.