Sc 43928

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.

For transient transfection, melanoma cells (18 × 10⁵ cells) were transfected with either 40pMol Mfn2-targeted siRNA (sc-43928, Santa Cruz or KH18422P, Qiagen) or with non-specific siRNA (sc-37007). Transfections were performed in Opti-MEM (Invitrogen) using Lipofectamine plus (Invitrogen) following the manufacturer's instructions. For stable Mfn2 knockdown, A375 melanoma cells were transfected under the same condition using different plasmids carrying respective shRNA or non-specific shRNA sequences (see Supplementary Material and Methods).