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. 2020 Feb 13;10:2558. doi: 10.1038/s41598-020-59517-y

Figure 5.

Figure 5

Deletion of the MeR2 enhancer by CRISPR/Cas9 system compromises the effect of COX-1 on Mbnl1 expression. The MeR2 enhancer in C2C12 myoblasts was knocked out (KO) using CRISPR/Cas9 system. (a) Schematic diagram of the target location and sequences of the two sgRNAs (sgRNA-1 and sgRNA-2) designed to delete MeR2. The protospacer-adjacent motifs (PAM) are marked in green and the target sequences of the sgRNAs are in red and blue, respectively. (b) PCR analysis and Sanger sequencing analysis showing the deletion of MeR2 in MeR2-KO C2C12 cells. The sequence of the non-edited cells (WT) is shown above. (c) The effect of knockdown of COX-1 and COX-2 on Mbnl1 mRNA expression in WT C2C12 cells and MeR2-KO C2C12 cells. The cells were treated with siRNA against COX-1 (siCOX1-a or siCOX1-b), COX-2 (siCOX2-a or siCOX2-b), both COX-1 and COX-2 (siCOX1a + 2a or siCOX1b + 2b), or control siRNA (siCont). Real-time RT-PCR analysis was performed 48 h after transfection. Expression levels of Mbnl1 mRNA are normalized to that of Gapdh, and also to the siCont-treated WT C2C12 cells. Mean and SD (n = 3 culture dishes) are indicated with individual values in red dots. **p < 0.01 and ***p < 0.001 compared to siCont by Student’s t-test with Bonferroni multiple comparison correction.