Additional file 1 of Inhibition of miR-199a-5p rejuvenates aged mesenchymal stem cells derived from patients with idiopathic pulmonary fibrosis and improves their therapeutic efficacy in experimental pulmonary fibrosis
Creators: name: Shi, Linli
name: Han, Qian
name: Hong, Yimei
name: Li, Weifeng
name: Gong, Gencheng
name: Cui, Jiangyu
name: Mao, Mengmeng
name: Liang, Xiaoting
name: Hu, Bei
name: Li, Xin
name: Luo, Qun
name: Zhang, Yuelin
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Marine Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. The plasmid Structure of Sirt1 and anti-miR-199a-5p which contain GFP reporter gene. (A) The plasmid Structure of Sirt1 which contains GFP reporter gene. (B) The plasmid Structure of anti-miR-199a-5p which contains GFP reporter gene. Figure S2. miR-199a-5p regulated the proliferation of MSCs. The level of miR-199a-5p in control-MSCs treated with miR control or miR-199a- 5p mimic was measured. (B) Immunostaining of the proliferation marker Ki67 and quantitative analysis of Ki67 positive cells in control-MSCs treated with miR control or miR-199a-5p mimic. Scale bar=100μm. (C) The level of miR-199a-5p in IPF-MSCs treated with miR control or miR-199a- 5p inhibitor was measured. (D) Immunostaining of the proliferation marker Ki67 and quantitative analysis of Ki67 positive cells in IPF-MSCs treated with miR control or miR-199a-5p inhibitor. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. Scale bar=100μm. **p &lt;0 .01; ***p &lt;0 .001. Figure S3. miR-199a-5p regulated the proliferation of MSCs by regulating autophagy. Representative images of autophagosomes examined using a TEM and quantitative analysis of autophagosomes in control-MSCs and IPF-MSCs. Scale bar=1μm. (B) Western blotting analysis of p62, Beclin, and LC3II/I protein expression in control-MSCs and IPF-MSCs. (C) Immunostaining of the proliferation marker Ki67 and quantitative analysis of Ki67 positive cells in control-MSCs transfected with miR control, miR-199a-5p mimic, or miR-199a-5p mimic + rapamycin. Scale bar=100μm. (D)Immunostaining of the proliferation marker Ki67 and quantitative analysis of Ki67 positive cells in lPF-MSCs transfected with miR control, miR-199a-5p inhibitor, or miR-199a-5p inhibitor + 3MA. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. Scale bar=100μm. n = 3. **p &lt;0 .01; ***p &lt;0 .001. Figure S4. The 3′UTR of Sirt1 contains one binding site for miR-199a-5p. (A) Predicted binding site for miR-199a-5p within the 3′UTR sequence of Sirt1. (B) Western blotting analysis of the expression level of Sirt1 in control-MSCs treated with miR control, miR-199a-5p mimic. (C) The luciferase reporter vector containing WT Sirt1 3′UTR or mutant 3′UTR was cotransfected with miR-199a-5p mimic or miRNA control into HEK293 cells. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. ***p &lt;0.001; ns: not significant.