primary human skeletal myoblasts  (PromoCell)

Journal: Molecular therapy. Nucleic acids

Article Title: miR-539-5p regulates Srebf1 transcription in the skeletal muscle of diabetic mice by targeting DNA methyltransferase 3b.

doi: 10.1016/j.omtn.2022.08.013

Figure Lengend Snippet: Figure 2. miR-539-5p inhibits Dnmt3b levels by binding to its 30 UTR (A) Differentiated C2C12 cells were transfected with the scramble (Scr) or mimics of miR-539-5p, miR-381-3p, or miR-31-5p (1–50 nM). Upon termination of incubation at 48 h, cells were lysed, and 40 mg protein was subjected to western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analyses of the blots are shown below. (B) Differentiated C2C12 cells were transfected with the Scr or the miR-539-5p mimic with or without its inhibitor. After 48 h, transcript levels of Dnmt3b were quantified by qRT-PCR. 18S rRNA was taken as the loading control. (C) Cells incubated as in (B) were lysed, and lysates (40 mg) were assessed for DNMT3b protein levels by western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analysis of the blots is shown below. (D) Depiction of the miR-539-5p binding site in the 30 UTR of Dnmt3b and the mutations (red) incorporated in the miRNA binding site of the Dnmt3b 30 UTR (MT) as described under “Materials and methods.” (E) C2C12 cells were transfected with wild-type (WT) or mutated (MT) 30 UTR luciferase constructs of Dnmt3b together with the Scr or the miR-539-5p mimic with or without its inhibitor. After 48 h of incubation, cells were harvested, and luciferase activity was measured as described under “Materials and methods.”. Renilla luciferase activity was normalized to firefly luciferase activity. (F) C2C12 cells were transfected with biotin-labeled Scr or biotin-labeled miR-539-5p mimic (50 nM), and after 48 h, cells were harvested, lysed, and pulled down using streptavidin-linked Dynabeads. Enrichment of Dnmt3b mRNA in biotin-labeled Scr- or miR-539-5p mimic- transfected cells was quantified by real-time PCR using Dnmt3b-specific primers. (G) Primary human skeletal muscle cells were transfected with the Scr or the miR-539-5p mimic with or without its inhibitor. Upon termination of incubation (48 h), DNMT3b expression was assessed by western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analysis of the same is given alongside the blot. All experiments were performed in at least 3 sets for each group. (H and I) 1 mg RNA from skeletal muscle of chow diet- and HFD-fed mice (n = 5) was reverse transcribed and subjected to qRT-PCR to evaluate the expression of miR-539-5p (H) and Dnmt3b (I). Sno234 and 18S rRNA, respectively, were used as normalization controls. Values are means ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: For validation in primary cells, human primary myoblast cells (PromoCell, Germany) were cultured in 12-well plates (CellBIND, Corning, NY, USA) in skeletal muscle growth medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Binding Assay, Transfection, Incubation, Western Blot, Control, Quantitative RT-PCR, Luciferase, Construct, Activity Assay, Labeling, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription