Journal: Aging Cell
Article Title: Age‐related changes in miR‐143‐3p:Igfbp5 interactions affect muscle regeneration
Figure Lengend Snippet: miR‐143 represses Igfbp5 expression in mouse and human primary myoblasts. (A) Alignment of putative miR‐143 target site in the 3′ UTR of Igfbp5 gene; human and mouse sequences are indicated; conserved miR‐143 putative target site is indicated in grey; complementary nucleotides are shown in grey, and miR‐143 seed sequence is highlighted. (B, C) Endogenous Igfbp5 protein and mRNA expression is regulated by miR‐143 in the mouse and human myoblasts, as shown by Western blot or qPCR , respectively. (D, E) GFP ‐Igfbp5 3′ UTR sensor constructs containing conserved mouse wild‐type or mutated miR‐143 target site were transfected into mouse myoblasts. Co‐transfection with miR‐143 mimic (miR‐143), but not miR‐24 mimic (not predicted to target Igfbp5), led to the downregulation of GFP protein expression compared to mock‐transfected control (Ctrl), as shown by Western blot. Point mutations in the micro RNA target site (mutant) rendered the sensor construct unresponsive. (F, G) Western blot showing increased expression of IGFBP 5 protein in FACS ‐sorted satellite cells from the old mice compared with the satellite cells from the adult mice. qPCR data show SEM ; * – P
Article Snippet: Transfections Myoblasts were transfected with 100 nm miRNA‐143‐3p or anti‐miR or 1 μg Igfbp5 overexpression vector that did not contain 3′UTR (Addgene, deposited by Johnson lab, University of California, San Francisco, CA, USA, Washington University), using Lipofectamine 2000™ , Life Technologies, Paisley, UK (Goljanek‐Whysall et al ., ).
Techniques: Expressing, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Construct, Transfection, Cotransfection, Mutagenesis, FACS, Mouse Assay