Allstars mm rn cell death control sirna

Four different of siRNA sequences, Mm-Pthr1-5, Mm-Pthr1-6, Mm-Pthr1-7, and Mm-Pthr1-8 (Qiagen, Hilden, Germany), were used to inhibit the expression of PTH1R in ZHTc6-MyoD cells during differentiation. AllStars Mm/Rn Cell Death Control siRNA (Qiagen) was used for the cell death control, and AllStars Neg siRNA AF488 (Qiagen) was used for a negative control. The differentiation of the ZHTc6-MyoD cells was induced 2 days before transfection. A total of 5 × 10⁵ cells were seeded in a 6-well type I collagen-coated plate. The cells were incubated for a short time and transfected with 3000 ng siRNA using the HiPerFect transfection reagent (Qiagen), according to the manufacturer's instructions. The differentiation medium was replenished at 48 h post-transfection.

Atorvastatin and lithium chloride (LiCl) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies targeting GSK-3β, Caspase-9 and B-cell lymphoma 2 (Bcl-2) were provided by BD Biosciences (Franklin Lakes, NJ, USA). Antibody targeting Cytochrome c (Cyt c) was from Abcam (Cambridge, MA, USA). Antibodies targeting α-actin, β-actin and GAPDH were provided by Sigma-Aldrich; Merck KGaA. LY294002 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The syn-rno-miR-199a-5p miScript miR mimic, anti-rno-miR-199a-5p miScript miR inhibitor, AllStars negative control small interfering (si)RNA, AllStars Mm/Rn cell death control siRNA, and HiPerFect transfection reagent were purchased from Qiagen, Inc. (Valencia, CA, USA) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega Corporation (Madison, WI, USA).

The H9c2 cells at a density of 2×10⁵ cells/well were seeded in 6-well plates for 12 h. Prior to transfection, the medium was replaced with DMEM without serum and antibiotics. The AllStars Mm/Rn Cell Death Control siRNA (Qiagen, Inc.) at 10, 20 and 50 nM were added and incubated for 24, 48, and 72 h to determine the optimal conditions. Transfection efficiency was observed and evaluated under a light microscope (Olympus Corporation, Tokyo, Japan). Transfection with 20 nM was selected due to the highest efficiency, which was >70%. HiPerFect transfection reagent (Qiagen, Inc.) was used as a transfection reagent. Syn-rno-miR-199a-5p miScript miR mimic and anti-rno-miR-199a-5p miScript miR inhibitor were used (20 nM) to respectively overexpress and inhibit the expression of miR-199a-5p. AllStars negative control siRNA was used as a negative control.