Sc 61556

The shRNA-targeting SIRT3 (mouse, sc-61556) and scrambled RNA (mouse, sc-108060), and shRNA transfection reagent (mouse, sc-108061) were purchased from Santa Cruz Biotechnology. AML12 cells were transfected with 2 μg shRNA for 6 h according to the manufacturer's protocol. Cells were switched to fresh medium and incubated for an additional 24 h. Then, cells were selected with 2 μg/ml puromycin for 6 days, and then 4 μg/ml puromycin for 6 days. Thereafter, cells were pooled together for further experiments.

The pcDNA3.1-SIRT3 plasmid was generated in our previous study [45 (link)]. Briefly, 3T3-L1 cells (4 × 10⁵) were seeded in 35 mm plates and cultured for 24 h. The cells were transfected with 10 μg plasmids (pcDNA3.1 or pcDNA3.1-SIRT3) using Lipofectamine 3000 reagent. 24 h after transfection, 800 μg/mL G418 was added to select positive cells for 2 weeks.
3T3-L1 cells at 50% confluence were transfected with 4 μg shRNA (shRNA targeting SIRT3 (mouse, sc-61556) or scrambled shRNA (mouse, sc-108060, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 6 h according to the manufacturer’s protocol. After incubated with fresh medium for an additional 48 h, cells were selected with 10 μg/mL puromycin for two weeks. Thereafter, cells were pooled together for further experiments.