Fig 2: FFV Promoted Cardiac Reprogramming by Functioning during the Late Phase(A) GMT-transduced MEFs were cultured with either FBS (black bars, left) or FFV (red bars) as indicated for 4 weeks. The number of beating cells in three independent triplicate experiments is shown (right).(B and C) FACS analyses for aMHC-GFP and cTNT expression in GMT-transduced MEFs cultured with FBS or FFV for 1 week. Quantitative data are shown in (C) (n = 3 independent triplicate experiments).(D) qRT-PCR for Mesp1 and isl1 expression in GMT-transduced cells cultured with FBS or FFV (n = 3 independent triplicate experiments).(E) qRT-PCR for Mesp1, isl1, Tnnt2, and Tnni3 expression in MEFs, iCMs, mouse ES-derived CPCs, and hearts (n = 3 independent triplicate experiments). The iCMs expressed cardiac genes, but not CPC genes.(F) Schematic representation of EdU treatment during cardiac reprogramming.(G) FACS analysis of EdU incorporation in mock-transduced cells and GMT-transduced MEFs cultured with FBS or FFV after 4 weeks of transduction.(H) Immunocytochemistry for cTNT in mock-transduced and GMT-transduced MEFs cultured with FBS or FFV for 4 weeks, coupled with 2 weeks of EdU incorporation. The majority of immunopositive cells were negative for EdU in the iCMs.(I) Quantitative data of immunopositive cells for HCN4, MLC2a, and MLC2v in iCMs cultured with FBS or FFV for 4 weeks (n = 7 independent triplicate experiments).(J) GMT-transduced cells were cultured with FBS for 1 week and then split into either FBS or FFV groups (left). The number of beating cells per well on 24-well plates is shown (right, n = 3 independent triplicate experiments).(K) GMT-transduced cells were cultured with FBS for 2 weeks, and the media were changed to FFV or IFFV (FFV + IWP4). The number of beating cells after 4 weeks is shown (n = 3 independent triplicate experiments).All data are presented as mean ± SD. **p < 0.01; *p < 0.05 versus the relevant control. ns, not significant. Scale bars represent 100 µm.See also Figure S2.