Dharmafect 1 transfection reagent

Sirt1 siRNA 15 nM (Santa Cruz Biotechnologies), Sirt2 siRNA 30 nM (Thermo Fisher Scientific), or Sirt3 siRNA 30 nM (Thermo Fisher Scientific) was transfected into a HeLa cell line with DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Corresponding concentrations of control siRNA were used as negative controls. Following transfection, cells were then maintained in a humidified 37 °C incubator with 5% CO₂ for another 48 hr (for Sirt1 and Sirt2) or 72 hr (for Sirt3).

RCC4 and HCT116 cell lines were cultured in DMEM (Hyclone) with 10% FBS and 1% pen‐strep (P/S, Hyclone). SKOV3ip.1 cells were cultured as described previously.¹³ RCC4 and HCT116 cell lines were kindly provided by Amato Giaccia Lab (Stanford University). For short‐term KDM4B knockdown experiments, cells were transiently transfected with siRNA (Dharmacon siGenome Smartpool) specifically targeting KDM4B (siK4B, Thermo Fisher Scientific) using DharmaFECT 1 transfection reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Scrambled siRNA (Dharmacon siControl 2) was used as the control group (siCon, Thermo Fisher Scientific). For hypoxia treatment, cells were cultured for 16 h in Ruskinn InVivo300 glove‐box hypoxic incubators (Baker), where oxygen levels were set at either 2% or 0.5%.

To investigate the effect of miR-33b, we transfected a commercially available mature miRNA molecule, Pre-miR™ miRNA Precursor (hsa-miR-33b) (Ambion, Austin, TX, USA), into confluent cells. The transfectant hsa-miR-33b is not a hairpin construct but a mature miRNA molecule, so that although it was designed for use with human cells, it can be applicable to pigs, cattle, or dogs, because the mature miRNA sequence of miR-33b is 100 % identical among these species. For the miR-NC in experimental group 2, we used Pre-miR™ Negative Control #1, which is a Pre-miR™ molecule designed to produce no identifiable effects on known miRNA function (Ambion). These transfectants were transfected by using the DharmaFECT 1 transfection reagent according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). Four replicate samples of cells were harvested at 2, 4, 8, 12, and 16 days after transfection and induction of adipocyte differentiation.

For transient silencing of endogenous MAD2 in 8505c and BCPAP cells, TriFECTa RNAi Kit (Integrated DNA Technologies Inc, Coralville, IA, USA) was used following manufacturer’s instructions. A duplex targeting a site absent in human genome was used as ‘universal’ negative control (NC). Three different siRNA oligonucleotides (siRNA1, siRNA2 and siRNA3) were transfected at 1 nM concentration using DharmaFECT 1 Transfection reagent (Thermo Scientific Inc, Waltham, MA, USA), according to manufacturer’s instructions. The day before transfection, cells were plated in antibiotics-free medium. Cells were harvested 72 h after transfection and gene-silencing efficiency was evaluated by protein levels analysis.

For transient silencing of endogenous METTL3, TriFECTa RNAi Kit (Integrated DNA Technologies Inc, Coralville, IA, USA) was used. A “universal” negative control duplex, which targets a site absent in the human genome, was used. Two different siRNA oligonucleotides (siRNA 1 and siRNA 2) were transfected at a concentration of 5 nM using DharmaFECT 1 Transfection reagent (ThermoFisher Scientific), according to the manufacturer’s instructions. The day before transfection, SW1736 and 8505C cells were plated in an antibiotic-free medium. Cells were harvested 48 h after transfection, and gene-silencing efficiency was evaluated by protein level analysis.