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Figure 5


Fig. 5. Effect of shRNA targeting {alpha}-SMA in ES cells. (A) Proteins of undifferentiated cells (lanes 1 and 4, untransduced ES cells; lanes 2 and 5, ES cells transduced with lentivectors expressing shCont; lanes 3 and 6, ES cells transduced with lentivectors expressing shSMA3) were submitted to SDS-PAGE (Coomassie Blue, lanes 1-3), transferred onto nitrocellulose and blotted with anti-Oct-4, a marker of pluripotenciality (lanes 4-6). (B) Cell cycle analysis by FACS cytometry of propidium iodure labeled untransduced (top), shCont (middle) and shSMA3 (bottom) ESC. (C) Proteins of EBs at day 6 (lanes 1 and 4, untransduced ESC; lanes 2 and 5, transduced with lentivectors expressing shCont; lanes 3 and 6, ES cells transduced with lentivectors expressing shSMA3) were submitted to SDS-PAGE (lanes 1-3; Coomassie Blue staining) and immunoblotted with anti-{alpha}-SMA, anti-{alpha}-SKA and anti-beta-cytoplasmic actin antibodies (lanes 4-6). (D) The percentage of beating EBs at day 8 was assessed under all three conditions. Bars represent s.e.m. of five independent experiments (**P<= 0.001). (E) Impact of {alpha}-SMA downregulation on the expression of cardiac transcription factors Nkx2.5 (black bars) and MEF2C (white bars) was evaluated by real-time RT-PCR. Bars represent s.e.m. of three independent experiments.*P<=0.05, **P<=0.001.





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