Fig 1: miR‐590 directly represses Sp1 expression. qPCR analysis of relative Sp1 mRNA expression in porcine cardiac fibroblasts and human cardiac fibroblasts (A), treated with GFP, GMT, miR‐590 and GMT plus miR‐590 (n=3). *P<0.05 vs relevant control (Kruskal–Wallis test). B, Western blot analyses for Sp1 expression in porcine cardiac fibroblasts treated with GFP, GMT, miR‐590 and GMT plus miR‐590 (n=3). C, qPCR analysis of relative Sp1 mRNA expression in porcine cardiac fibroblasts transfected with control (scrambled) and anti‐miR590 (n=3; Wilcoxon rank‐sum test). All data are presented as mean±SEM. *P<0.05 vs relevant control. GFP indicates green fluorescent protein; miR, microRNA; qPCR, quantitative polymerase reaction; Sp1, specificity protein 1.
Fig 2: Overexpression of Sp1 disrupts cardiac reprogramming in GMT/miR‐590‐transduced cells. A, Relative mRNA expression of cardiomyocyte marker genes (TNNT2, MYH6) in porcine cardiac fibroblasts transduced with GMT and/or miR‐590, and with or without Sp1 overexpression induced by Sp1 cDNA (n=3). *P<0.05 versus relevant control (Kruskal–Wallis test). B, Relative mRNA expression of fibroblast marker genes (COL1A1 and COL3A1) in porcine cardiac fibroblasts transduced with transduced with GMT and/or miR‐590, and with or without Sp1 overexpression induced by Sp1 cDNA (n=3). *P<0.05 versus relevant control (Kruskal–Wallis test). C, Left: Representative flow cytometry plots for analyses of cTnT + cells 2 weeks after transduction of porcine cardiac fibroblasts with GMT with or without miR‐590 transduction, and with Sp1 overexpression. Right: Summary of flow cytometry analyses. Percentage of cTnT + cells following infection of porcine cardiac fibroblasts (n=3). All data are presented as mean±SEM. *P<0.05 versus GFP; # P<0.05 versus GMT/miR‐590 (Wilcoxon rank‐sum test). COL indicates collagen; cTnT, cardiac troponin T; GFP, green fluorescent protein; miR, microRNA; Sp1, specificity protein 1.
Fig 3: miR‐590‐mediated Sp1 is critical for cardiac reprogramming. A, Relative mRNA expression of Sp1 in untreated (mock) porcine cardiac fibroblasts and porcine cardiac fibroblasts transfected with scrambled siRNA or anti‐Sp1 siRNA (1, 5, and 10 μmol/L; n=3). *P<0.05 versus mock (Wilcoxon rank‐sum test). B, Relative mRNA expression of cardiomyocyte marker gene (TNNT2, MYH6) in and porcine cardiac fibroblasts transduced with GMT and/or miR‐590, and with or without si‐Sp1 (n=3). *P<0.05 versus relevant control (Kruskal–Wallis test). C, Relative mRNA expression of fibroblast marker genes (COL1A1 and COL3A1) in, and porcine cardiac fibroblasts transduced with, GMT and/or miR‐590, and with or without si‐Sp1 (n=3). *P<0.05 versus relevant control (Kruskal–Wallis test). D, Left: Representative flow cytometry plots for analyses of cTnT + cells 2 weeks after transduction of porcine cardiac fibroblasts with GMT and si‐Sp1 or miR‐590. Left to right: GFP, GMT, GMT plus miR‐590, and GMT plus si‐Sp1. Right: Summary of flow cytometry analyses. Percentage of cTnT + cells following infection of porcine cardiac fibroblasts (n=3; Wilcoxon rank‐sum test). Data are presented as mean±SEM. *P<0.05 versus relevant control. COL indicates collagen; cTnT, cardiac troponin T; GFP, green fluorescent protein; miR, microRNA; Sp1, specificity protein 1.
Supplier Page from Sino Biological, Inc. for Human SP1 Gene ORF cDNA clone in cloning vector