Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

doi: 10.1016/j.omtm.2020.05.006

Figure Lengend Snippet: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs (A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O 2 ) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, α-tubulin served as a loading control.

Article Snippet: AML12 mouse hepatocytes (cat. #CRL-2254; ATCC, Manassas, VA, USA) were cultured in DMEM/F12 medium supplemented with 10% FBS and Insulin -Transferrin-Selenium (ITS suplement; ATCC): insulin (0.005 mg/mL), transferrin (0.005 mg/mL), selenium (5 ng/mL), and with dexamethasone (40 ng/mL) in the presence of antibiotics.

Techniques: Expressing, Western Blot, Transduction, Positive Control, Isolation, Transfection, Plasmid Preparation, Control