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Fig. 1 Mesoderm induction and vascular cell differentiation from hiPSCs. Undifferentiated hiPSCs were sequentially induced to differentiate toward mesoderm cells, ECs and SMCs, respectively. Cells were harvested at various differentiation stages (undifferentiated hiPSCs, day 1 differentiating hiPSCs treated with ROCK inhibitor (RI), day 4 differentiating hiPSCs (end of mesoderm differentiation stage, MESO), and day 8 differentiating hiPSCs (4 days of SMC or EC differentiation)) and subjected to RT-qPCR (A-C), Western blot (D), and immunofluorescence (IF) staining (E) analysis, respectively. (A-C) RT-qPCR analysis showing decreased expression levels of pluripotency genes (A), but increased expression levels of mesodermal (Brachyury/T) (B), as well as EC and SMC (C) genes, during mesoderm induction and vascular differentiation from hiPSCs. (D) Western blot detection of protein expression. Representative (top) and semi-quantitative data (bottom) of protein expression levels were presented here. Numbers represent relative protein expression levels against α-tubulin as indicated, which is averaged from three independent experiments. Samples with ‘0’ indicate that this protein was undetectable in that sample from all three experiments. (E) IF staining analysis of cellular marker protein expression in hiPSC-derived ECs (up panels) and SMCs (bottom panels) at day 8. The data presented here are representative (D & E) or mean±S.E.M. (A-C) of three (D & E) or five (A-C) independent experiments (n=3 or 5). *P<0.05 (vs hiPSCs).