Ith BSA-conjugated PA (100 ) for 24 h in GM before differentiation as described previously [26]. Unless otherwise stated, all reagents and components were purchased from Sigma-Aldrich (St. Louis, MO, USA). two.2. Transfection of Oligonucleotides C2C12 myoblasts were transfected with scrambled handle RNA (scRNA), CFL2 siRNA (siCFL2), miR-325-3p mimic, or antimiR-325-3p (an inhibitor of miR-325-3p; a two -O-methylmodified antisense oligonucleotide against mature miR-325-3p) from Genolution (Seoul, Korea) at final concentrations of 200 nM within a GM working with Lipofectamine 2000. The sequences from the oligonucleotides employed for this study are listed in Table S1. two.3. RNA Extraction, PCR and Quantitative Real-Time PCR (qRT-PCR) Total RNA of C2C12 cells was extracted employing a Qiazol reagent and miRNeasy Mini Kit (Qiagen, Hilden, Germany) 24 h following transfection. The good quality and concentration of RNAs have been assessed by gel electrophoresis along with a UV-1700 PharmaSpec spectrophotometerCells 2021, ten,three of(AICAR custom synthesis Shimadzu, Kyoto, Japan). The RNA was then reverse-transcribed working with the miScript II RT Kit (Qiagen). To figure out mRNA and miRNA expression levels, qRT-PCR and RT-PCR were performed utilizing specified primers, SYBR Green I, and iTaq polymerase (Promega, Bioactive Compound Library Purity Medison, WI, USA) in conjunction having a Light-Cycler 480 (Roche Applied Science, Penzberg, Germany). Specifics in the primers applied for RT-PCR and qRT-PCR and reaction conditions are described in Table S2. The 2-Ct method was employed to calculate relative mRNA expressions, and final results have been normalized by U6 snRNA expression. 2.four. Dual-Luciferase Assay A segment in the murine CFL2 three UTR (358-nt long) containing the possible miR-3253p binding web-site (CFL2wt) was chemically synthesized by RT-PCR applying distinct primers listed in Table S2. The wild-type reporter construct was created by subcloning CFL2wt into the pmirGLO vector (Promega) utilizing SacI and XbaI websites. Mutation from the miR-325-3pbinding site (CFL2mut) was synthesized by PCR-based site-directed mutagenesis applying overlapping oligonucleotides (Table S2). For dual-luciferase target validation assays, C2C12 cells were plated inside a 12-well plate at a density of 1 105 cells/well. After 24 h, a pmirGLO vector containing CFL2wt or CFL2mut was co-transfected with scRNA or miR-325-3p mimic into cells applying Lipofectamine 2000. The Dual-Luciferase Reporter Assay System 100 Kit (Abcam, Cambridge, UK) was utilized to determine luciferase activities 24 h soon after transfection as described recently [27]. two.5. Immunoblot Analysis For protein preparation, C2C12 cells were collected by centrifugation and lysed employing PBS containing 2 Triton X-100 and 1 phosphatase inhibitor cocktail II (Abcam) as previously described [28]. The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were employed for nuclear and cytoplasmic protein fractionation. Protein concentrations had been determined utilizing the Bradford technique, and then cell lysates have been mixed with 2X Laemmli buffer and boiled for 10 min at one hundred C. Proteins have been separated by SDS-PAGE at 20 of protein/lane and transferred to nitrocellulose membranes (Amersham, Germany). The membranes were then blocked with five skim milk in TTBS (0.5 TBS-Tween 20 in PBS) for 1 h and incubated with precise primary antibodies at four C overnight (Table S3). The membranes were washed with TTBS 5 times and incubated having a secondary antibody for 1 h. Lastly, protein bands have been visualized utilizing a Femto reagent (Thermo Fisher Scientific) with Fusion.