Ative binding web sites of miR-325-3p Figure MiR-325-3p regulated CFL2 expression by binding towards the three UTR of CFL2. (A) Putative binding web sites of miR-325on the 3the 3UTR fragments of CFL2 mRNA. (B) Sequence alignment miR-325-3p binding web page with wild-type (CFL2wt) or 3p on UTR fragments of CFL2 mRNA. (B) Sequence alignment of of miR-325-3p binding web site with wild-type (CFL2wt) or mutant (CFL2mut) 3UTR of CFL2. (C) MiR-325-3p mimic or scrambled control mutant (CFL2mut) three UTR of CFL2. (C) MiR-325-3p mimic or scrambled manage RNA (scRNA) had been co-transfected with (scRNA) have been co-transfected having a dual-luciferase reporterconstruct containing CFL2wt or CFL2mut in C2C12 cells, and relative luciferase activity was construct containing CFL2wt or CFL2mut in C2C12 cells, and relative luciferase activity was a dual-luciferase reporter measured 24 right after transfection. (D) CFL2 protein levels have been analyzed 24 h just after transfection with 200 nM scRNA measured 24 h h soon after transfection. (D) CFL2 protein levels had been analyzed 24 h right after transfection with 200 nM ofof scRNA control or miR-325-3p mimic by immunoblotting. (E) The mRNA expressions had been determined by RT-PCR (upper panel) control or miR-325-3p mimic by immunoblotting. (E) The mRNA expressions were determined by RT-PCR (upper panel) and qRT-PCR (reduced panel). Immunoblot and qRT-PCR results are shown as relative ratios versus scRNA manage. All and qRT-PCR (reduce panel). indicates SEMsand qRT-PCR resultssignificance arerelative ratios versus scRNA handle.vs. final results are presented TPA-023B Autophagy because the Immunoblot (n 3), and levels of are shown as presented as , p 0.01; , p 0.001 All benefits are presented because the means SEMs (n three), and levels of significance are presented as , p 0.01; , p 0.001 vs. scRNA controls. scRNA controls.3.3. MiR-325-3p Increased F-Actin and Nuclear Yes-Associated Protein (YAP) 3.3. MiR-325-3p Improved F-Actin and Nuclear Yes-Associated Protein (YAP) In a previous study, knockdown of CFL2 provoked the accumulation of F-actin in In a prior study, knockdown of CFL2 provoked the accumulation of F-actin in myoblasts [25], and therefore, we hypothesized that miR-325-3p increases F-actin by inhibiting myoblasts [25], and as a result, we hypothesized that miR-325-3p increases F-actin by inhibitCFL2 expression in myoblasts. Transfection of myoblasts with siCFL2 significantly deing CFL2 expression in myoblasts. Transfection of myoblasts with siCFL2 substantially creased CFL2 level by 60 (Figure 3A) and transfection with miR-325-3p mimic effidecreased CFL2 level by 60 (Figure 3A) and transfection with miR-325-3p mimic effectively elevated (200-fold) the cellular degree of miR-325-3p in myoblasts (data not shown). ciently elevated (200-fold) the cellular level of miR-325-3p in myoblasts (information not shown). Beneath this experimental situation, miR-325-3p mimic or siCFL2 considerably enhanced Below this experimental condition, miR-325-3p mimic or siCFL2 substantially increased F-actin as determined with FITC-coupled phalloidin (Figure 3B). Since actin levels reF-actin as determined with FITC-coupled phalloidin (Figure 3B). Since actin levels reClovamide Purity & Documentation mained continual through differentiation irrespective of treatment options, the induction of F-actin mained constant in the course of differentiation no matter treatments, the induction ofdue to F-actin accumulation by miR-325-3p mimic was ascribed to lack of actin depolymerization accumulation by miR-325-3p mimic was ascribed to lack of actin depolymerization due CFL2 suppression. Recentl.
