Fig. 3

Employment of control and DMD iPSC in high-content screening setting for the assessment of TGFβ response. a Schematic representation of the protocol used for HCS. To ensure higher control over cell number and reproducibility, cells have to be dissociated in single cells before doxycycline treatment. Cells are plated in Matrigel-coated 384-optical well plates, fixed, and stained and then analyzed by MetaXpress Analysis software. b Representative immunostaining of control and DMD iPSC (ex59X)-derived myotubes, stained for pSMAD3 in normal condition and upon treatment with TGFβ (20 ng/ml) and c quantification of the percentage of pSMAD3 fluorescence obtained by MetaXpress Image analysis software, setting background and size parameters as described in the “Materials and methods” section. Data are expressed as fold change. d Gene expression of TGFβ1 in control and DMD iPSC-derived myotubes expressed as fold induction of TGFβ-treated versus untreated cells. e Gene expression of TGFβ1 mRNA expression in control and DMD iPSC-derived myotubes pre-treated with TGFβR1 inhibitor (SB-431542 (10 μM)), expressed as fold induction of TGFβ-treated versus untreated cells. All data are represented as average ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test)