FOXO1A protein expression during in vitro differentiation of HDSC and THESC. Cultures were incubated with cAMP and/or E2P4 for 3 and 9 days. Protein expression and localization of FOXO1A were determined by Western blotting and immunofluorescence, respectively, as described in Materials and Methods. A) Western blot detecting FOXO1A (78 kD) in total cellular extracts. Marker bands (kD) are depicted on the left side. GAPDH (37 kD) was used as a loading control. Representative examples are shown. B) Immunflourescence in untreated and cAMP-stimulated HDCS and THESC. Representative pictures at a 200 fold magnification are shown. C) Quantification of FOXO1A-positive nuclei in cAMP and/or E2P4-treated HDSC and THESC after immunofluorescence. Ratio (percentage) of positive nuclei were evaluated as described above. Bars represent mean values ± SEM of three experiments. * indicates p < 0.05 compared to negative control (n.c.) of the same day; n.s., not significant; # indicates p < 0.05 between cAMP-treated and cAMP/E2P4-stimulated cultures.