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. 2020 Jul 21;48(16):8927–8942. doi: 10.1093/nar/gkaa596

Figure 5.

Figure 5.

Dicer and the 3′UTR are required for regulation of MRTF-A and myogenic differentiation. (A) Transient knockdown of Dicer1 using siRNA. After 48 h of incubation, myogenic differentiation was induced in C2C12 when indicated. miR-1a-3p and miR-486-5p were quantified by qRT-PCR for validation (top right). The proteins MYH, MRTF-A, myogenin and tubulin were compared to a control transfection (siCtrl.) by immunoblotting (top left). Quantification of relative protein amounts is depicted below. Error bars, SD (n = 3) (B–D) The 3′UTR at the endogenous locus of MRTF-A was deleted using CRISPR-Cas9. Selected pools of C2C12 cells were analysed for protein expression and differentiation. (B) Immunoblot and quantification of MRTF-A protein level in cells with deleted 3′UTR (ΔUTR) compared to control cells lacking the sgRNA (Cas9) (C) Phase contrast images (10x). (D) Quantified myogenin and MYH protein expression, shown as percentage from the Cas9 control cells at d2, d4 and d6 of differentiation Error bars, SEM (n = 4); *P< 0.05, **P< 0.01, ***P< 0.001 (Student’s t-test). Size bar, 400 μm.