Fig. 2

Evaluation of the myogenic potential of DMD iPSC^BM and activation status of TGFβ signaling. a Immunofluorescence for MyHC, performed in cells differentiated into myotubes. Control and DMD iPSC (ex59X), expressing inducible ePB vectors for MyoD and BAF60C (iPSC^BM), were differentiated according to the protocol described in Fig. 1. Control and DMD human myoblasts were differentiated by medium switch; IMR90 cells, previously nucleofected with inducible ePB vector for MyoD (IMR90^MyoD), were differentiated by medium switch (see the “Materials and methods” section for details). To monitor the effect of the lack of DYSTROPHIN during myogenic conversion in IMR90, cells were transfected both in GM and DM conditions with siRNAs for DYSTROPHIN (siDMD) or a scrambled sequence (siScr). The level of DYSTROPHIN downregulation is shown on the left. b Myogenic index (bottom graph) was calculated by immunofluorescence staining, as the percentage of nuclei within MyHC-expressing myotubes. c Immunofluorescence for pSMAD in iPSC^BM, human myoblasts, and IMR90^MyoD-derived myotubes. Nuclei were visualized by DAPI. Scale bar, 50 μm. d The percentage of pSMAD3-positive cells was calculated counting 10 fields and using the same exposure parameters among the samples. e TGFβ1 relative expression monitored in all the cells. The expression levels are shown as relative to the control or siSCr. Data are represented as average ± SEM (n = 3) *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test)