Fig 1: Effects of p300 knockdown on human-skeletal myogenesis in MyoD-hiPSCs. (a) Schematic diagram showing transfection of MyoD-hiPSCs with siRNA against p300 (si-p300) at days 1 and 4. (b,c) MyoD-hiPSCs were transfected with si-p300 or negative control duplexes (Control). Whole-cell extracts were harvested at day 4 and 7 after myogenic induction, and p300 expression was evaluated using immunoblotting. p300 expression was quantified using Image J and normalised to ß-actin. n = 5. *p < 0.05, ***p < 0.001 versus control. Full-length blots are presented in Supplementary Figure S8. (d) Representative images of MyoD-hiPSCs at day 7 post-transfection with Control, si-p300, and miR-494 mimic. Scale bars: 100 µm. (e,f) The number (e) and length (f) of differentiated myotubes at day 7 was analysed in each microscopic field. n = 10 (e), n = 7 (f). ***p < 0.001 versus control. (g) Expression of myosin heavy chain 2 (MYH2), MYH7, and MYH1 in MyoD-hiPSCs transfected with Control, si-p300, or miR-494 mimic at day 7 after myogenic induction, as assessed via western blotting. Full-length blots are presented in Supplementary Figure S9. (h) The level of MYH2 was quantified using Image J and normalised to ß-actin. n = 5. *p < 0.05, **p < 0.01 versus control. (i) Expression of myogenin (MYOG) in MyoD-hiPSCs transfected with Control, si-p300, or miR-494 mimic at day 7 after myogenic induction, as assessed via western blotting. Full-length blots are presented in Supplementary Figure S10. (j) miR-494 expression in MyoD-hiPSCs transfected with Control and si-p300 at day 7 after myogenic induction, as analysed by Taqman RT-qPCR. Data were normalised with snRNA U6. n = 3.