Rg202560

Gene overexpression of Mfn1, Mfn2, Drp1, and Fis1 (RG207184; RG202218; RG202046; RG202560; Origene Technologies, Inc., Rockville, MD, USA) were performed using Lipofectamine® 2000 (1367620, Invitrogen) in the presence of Opti-MEM (31985-070, Gibco) for 18 h. A DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:1 was used for transfection. GFP-expression vector (OriGene-ORIPS100010, Origene) was used as mock control (Technologies, Inc., Rockville, MD, USA). For siRNA knockdown, interfering RNAs (sc-43927; sc-43928; sc-43732; sc-60643; Santa Cruz Biotechnology, Santa Cruz, CA) were delivered using Lipofectamine® 2000 (1367620, Invitrogen) in the presence of Opti-MEM (31985-070, Gibco) for 4 h, followed by washing and replacement with a fresh growth medium for 72 h. We grew cybrid cells individually under 25 mM glucose or glucose plus saturated fatty acid (FA) for 6 h to create different nutritional conditions. Saturated fatty acid palmitate (P9767; Sigma) was conjugated into bovine serum albumin as a carrier. The concentration of palmitate was 0.25 mM, similar to postprandial levels in humans (28 (link)). For drug treatment experiments, cells were treated with vehicle, 1 mM metformin (M0605000, Sigma-Aldrich), or 10 μM mdivi-1 (M0199, Sigma-Aldrich) for another 24 h.

Cells were starved of FBS for 16 h, followed by transfection of plasmid/siRNA or mdivi-1 treatment. Then, cells were stimulated with insulin (Sigma-Aldrich, St. Louis, MO, USA) at 0, 0.1, or 1 μM for 1 h and then collected for experimental assays. Gene overexpression and knockdown of Mfn1, Mfn2, Drp1, and Fis1 were performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Transfection of plasmid (RG207184; RG202218; RG202046; RG202560; Origene Technologies, Inc., Rockville, MD, USA) and siRNA (sc-43927; sc-43928; sc-43732; sc-60643; Santa Cruz Biotechnology, Santa Cruz, CA) spanned 24 and 48 h, respectively. Mock control of gene overexpression and siRNA was GFP-expression vector (OriGene-ORIPS100010, Origene Technologies, Inc., Rockville, MD, USA) and dsRNA with scramble sequence (sc-37007; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.