Fig. 3. Differential effect of SMA-FP and SKA-FP on beating and myofibril organization. (A) Schematic representation of the protocol used to treat EBs with FPs twice a day with 5 or 10 µg/ml of SMA-FP, or SKA-FP from days 6 to 8. At day 8, the capacity of the treated ES cells to differentiate into fully differentiated cardiomyocytes was assessed by determining the appearence of spontaneous beating. (B) Percentage of beating EBs at day 8 in untreated EBs (white bars), SMA-FP-treated (striped bars) and SKA-FP (black bars) treated EBs. Error bars represent s.e.m. of a total of five independent experiments (^**P0.001). (C) Western blot analysis of untreated EBs (lanes 1) and EBs treated with 10 µg/ml SMA-FP (lanes 2) or 10 µg/ml SKA-FP (lanes 3). Proteins were immunoblotted with anti-total actin (1C4), anti-SMA, anti-CAA and anti-SKA antibodies. (D) 3D reconstruction of confocal microscopy images of untreated EBs (a) and SMA-FP-treated EBs (b) fixed at day 8 and double-stained with anti- actinin (red) and anti-SMA (green) antibodies. (E) 3D reconstruction of confocal microscopy images of untreated (a) and SKA-FP treated (b) EBs fixed at day 8 and double-stained with anti- actinin (red) and anti-SKA (green) antibodies. Bars, 10 µm. Insets in b, magnification of the myofibrils.