Additional file 2 of Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation
Creators: name: Hu, Bowen
name: Zhao, Changbin
name: Pan, Xiangchun
name: Wei, Haohui
name: Mo, Guodong
name: Xian, Mingjian
name: Luo, Wen
name: Nie, Qinghua
name: Li, Hongmei
name: Zhang, Xiquan
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Environmental Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology

description: Additional file 2: Fig. S1 High GHR expression during myoblast differentiation. a The GHR expression profile in the CPM proliferation and differentiation phases. b Transfection of overexpression plasmid or siRNA during CPM differentiation. c Overexpression efficiency of plasmid was measured by EGFP fluorescence intensity at 48 h after transfection with pcDNA3.1-EGFP during the CPM differentiation; scaler bar, 200 μm. d Knockdown efficiency of siRNA was measured by Cy3 fluorescence intensity at 48 h after transfection with siNC-Cy3 during the CPM differentiation; scaler bar, 200 μm. Data are shown as mean ± SEM, **p &lt; 0.01. Fig. S2 GHR overexpression promotes mitochondrial biogenesis. a Overexpression efficiency was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. b The expression of genes involved in the GH-GHR-IGFs signaling pathway was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. c and d MTG staining of CPM was measured at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. White arrow labeled elongated myoblasts. Scaler bar, 25 μm. e and f Confocal images were observed at 48 h after co-transfection with pMitoTimer + pcDNA3.1-GHR and pMitoTimer + pcDNA3.1-GHR. Scaler bar, 10 μm. Green represents newly synthesized mitochondria, red represents mature mitochondria. Images were analyzed by Leica LAS X life science software. g The expression of genes involved in PGC1α-NRF1-TFAM signaling pathway was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. h The expression of mtDNA encoded genes was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. i The relative mtDNA content was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. j-l Western blots with anti-GHR, anti-PGC1α, anti-NRF1 and anti-β-actin at48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. Data are shownas mean ± SEM, *p &lt; 0.05, **p&lt; 0.01. Fig. S3 IGF1 overexpression promotes mitochondrial biogenesis. a Overexpression efficiency was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-IGF1and pcDNA3.1. b The expression of genes involved in the GH-GHR-IGFs signaling pathway was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. c and d MTG staining of CPM was measured at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. White arrow labeled elongated myoblasts. Scaler bar, 25 μm. e and f Confocal images were observed at 48 h after co-transfection with pMitoTimer + pcDNA3.1-IGF1 and pMitoTimer + pcDNA3.1-IGF1. Scaler bar, 10 μm. Green represents newly synthesized mitochondria, red represents mature mitochondria. Images were analyzed by Leica LAS X life science software. g The expression of genes involved in PGC1α-NRF1-TFAM signaling pathway was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. h The expression of mtDNA encoded genes was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. i The relative mtDNA content was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. j and k Western blots with anti-PGC1α, anti-NRF1 and anti-β-actin at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. Data are shown as mean ± SEM, *p &lt; 0.05, **p &lt; 0.01. Fig. S4 Local GHR regulates mitochondrial biogenesis via IGF1-PI3K/AKT/CREB signaling. a and b Western blots with anti-JAK2, anti-p-JAK2, anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 andanti-β-actin at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. c and d Western blots with anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. e The expression of CREB and PGC1α was measured by RT-qPCR at 48 h after transfection with si-CREB and si-NC. f and g Western blots with anti-PGC1α, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with si-CREB and si-NC. h Dual-Luciferase report assays of CREB knockdown co-transfected with reporter vectors containing different length of 5′ upstream region of PGC1α. Data are shown as mean ± SEM, *p &lt; 0.05, **p &lt; 0.01. Fig. S5 GHR or IGF1 overexpression enhances mitochondrial function. a ΔΨm was measured by the fluorescence of JC-1 at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. b ΔΨm was measured by the fluorescence of JC-1 at 48 h after transfectionwith pcDNA3.1-IGF1 and pcDNA3.1. c ATP level was measured at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. d Reactive oxygen species production was measured by the fluorescence of DCF at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. e ATP level was measured at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. f Reactive oxygen species production was measured by the fluorescence of DCF at 48 h after transfection with pcDNA3.1-IGF1 and pcDNA3.1. Data are shown as mean ± SEM, *p &lt; 0.05, **p&lt; 0.01. Fig. S6 GHR overexpression represses myoblast differentiation. a CCK-8 assays were performed after transfection with pcDNA3.1-GHR and pcDNA3.1. b and c EdU proliferation assays were performed after transfection with pcDNA3.1-GHR and pcDNA3.1. d Cell cycle analysis were performed after transfection with pcDNA3.1-GHR and pcDNA3.1. e The expression of cell proliferation marker genes was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. f-h MyHC staining, myotube area and myoblast fusion index were measured at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. i The expression of myoblast differentiation marker genes was measured by RT-qPCR at 48 h after transfection with pcDNA3.1-GHR and pcDNA3.1. Data are shown as mean ± SEM, *p &lt; 0.05, **p &lt; 0.01.