Fig. 7: Phenotypes induced by FTOrs9939609-A are reversed by IGF2 inhibition and H19 knockdown. | Nature Communications

Fig. 7: Phenotypes induced by FTOrs9939609-A are reversed by IGF2 inhibition and H19 knockdown.

From: An obesogenic FTO allele causes accelerated development, growth and insulin resistance in human skeletal muscle cells

Fig. 7

a Left: Western blot of phospho-AKT, phospho-FOXO and GAPDH protein abundance in FTOrs9939609-TT-myotubes: H1, H7, H9 and FTOrs9939609-A-myotubes: FTO-H1-1, FTO-H1-2, FTO-H7-1, FTO-H7-2, FTO-H9-1, FTO-H9-2, with or without 0.8 µg/ml human IGF2 antibody inhibition for 7 days. Right: quantification of the abundance of insulin signaling proteins, n = 3 biologically independent samples. b Quantitative RT-PCR for myogenic markers in FTOrs9939609-TT-myotubes and FTOrs9939609-A-myotubes treated with or without 0.8 µg/ml human IGF2 antibody inhibition for 7 days, n = 3 biologically independent samples. c Quantification of fold change in FTOrs9939609-TT-myoblasts and FTOrs9939609-A-myoblasts numbers, with or without 0.8 µg/ml human IGF2 antibody inhibition for 7 days (FTO-TT vs. FTO-A, P = 0.02165), n = 3 biologically independent samples. d Left: representative images of senescent FTOrs9939609-TT-myoblasts and FTOrs9939609-A-myoblasts, with or without human IGF2 antibody inhibition for 7 days by passage 16. Senescent cells were stained for β-galactosidase (SA-βgal). Scale bars, 100 μm. Right: quantification of percentage of SA-βgal+ senescent FTOrs9939609-TT-myoblasts and FTOrs9939609A-myoblasts, n = 3 biologically independent samples. e Left: representative images of FoxO1-GFP+ nuclei numbers at the end of 14 days’ exposure to 1% HFD serum, with or without human IGF2 antibody inhibition. Scale bars, 200 μm. Right: quantification of the percentage of FoxO1-GFP+ nuclei numbers (FTO-TT vs. FTO-A, P = 0.01002), n = 3 biologically independent samples. f Quantitative RT-PCR for H19 and GAPDH (FTO-TT vs. FTO-A, P = 0.00136), n = 3 biologically independent samples. siNC: knockdown negative control; siH19: knockdown of H19 lncRNA. g Quantification of the abundance of myogenic and insulin signaling proteins, n = 3 biologically independent samples. siNC: knockdown negative control; siH19: knockdown of H19 lncRNA. H1 hESC line colored in purple, H7 hESC line colored in light blue and H9 hESC line colored in dark blue. Data are presented as mean + SEM. P values were calculated by two-tailed unpaired t-test. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.

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