Suppression of mTOR is required for highly efficient cardiac differentiation. A, The activity of mTOR signaling is inhibited by rapamycin administration, shown by the phosphorylation of p‐S6K1 (Thr389). B, The effects of mTOR knockdown and overactivation on cardiomyocyte differentiation. mTOR siRNA significantly decreases mTOR expression and activity. In contrast, TSC1 and TSC2 siRNA increase mTOR expression and activity. C, The percentage of cardiomyocytes following siRNA treatment, as measured by flow cytometry (n=4; **P<0.01). D and E, The dynamic fluctuation of mTOR activity in experiments without rapamycin treatment. mTOR activity was analyzed by Western blot; p70S6K1 (Thr389), S6K1, and β‐actin were used as the loading control (D). The transcription levels of mTOR signal‐associated genes including mTOR, 4E‐BP1, and p70S6K1 as measured by quantitative real‐time PCR (E). CHIR indicates CHIR99021; PCR, polymerase chain reaction; Rapa, rapamycin; si‐NC, negative control siRNA.