Trifecta dsirna kit

2.5 × 10⁵ THP-1 cells were transfected by using the HiPerFect Transfection reagent (#301705, Qiagen, Venlo, Netherlands) according to manufacturer’s instructions (protocol for suspension cell lines). Predesigned siRNA targeting human IRE1α, PERK, ATF6, CHOP and ATF3 were purchased from Integrated DNA Technologies (IDT, Coralville, USA) (TriFECTa DsiRNA Kit) (Table S1). For IRE1α and PERK, THP-1 cells were transfected before PMA differentiation and the treatment with FFAs performed 80 h post-transfection. For ATF6, PMA-differentiated THP-1 cells were transfected for 32 h before FFAs stimulation. CHOP and ATF3 siRNAs were transfected in PMA-differentiated THP-1 cells for 8 h before FFAs treatment.

PDXOs were transfected with siRNAs against MAPK8 and JUN (TriFECTa® DsiRNA Kit, Integrated DNA Technologies, IDT, Singapore) using Lipofectamine RNAiMAX (Thermo Fisher), or treated with JNK inhibitor SP600125 (Selleck Chem) and incubated overnight prior to treatment with ixazomib and dinaciclib for 24 hours. Cells were then pelleted and used for RNA and protein extraction as described above.

DsiRNA against specific genes were purchased from Integrated DNA Technologies as TriFECTa DsiRNA kit. DsiRNA mixed with DharmaFECT1 reagent were added to cells in 12-well plates in DMEM supplemented with 10% FBS and incubated at 37°C for 24 hours.

MDMs were transfected by using lipofectamine RNAiMax (13778, Invitrogen) according to manufacturer’s instructions. Predesigned siRNA targeting human HIF-1α, XBP-1s and IRE1α mRNA or control (CT) siRNA targeting any sequence were purchased from Integrated DNA Technologies (IDT, Coralville, USA) (TriFECTa DsiRNA Kit) (Table S2). MDMs were transfected with siRNA XBP1s and siRNA HIF-1α for 24h and siRNA IRE1α for 48h before SFAs or LPS stimulation.

To knock down endogenous YAP and TAZ levels, C2C12 cells were transfected with siRNAs against YAP (TriFECTa DsiRNA Kit, design ID, mm.Ri.Yap1.13, Integrated DNA Technologies, Inc.) or TAZ (Stealth siRNAs MSS227747, MSS227748, MSS227749, Thermo Fisher Scientific) using Lipofectamine 2000 (11668027, Thermo Fisher Scientific) according to the manufacturer's instructions. To analyze resulting protein expression levels, cells were lysed for Western blot analysis at indicated time points after transfection.
Cells were seeded in antibiotic‐free growth media for 24 h prior to transfection. Transfection solution was prepared as follows: 1 mg mL^–1 Lipofectamine 2000 was diluted to 30 µg mL^–1 in Opti‐MEM media (31985, Gibco) and mixed for 15 min, while 20 × 10⁻⁶m solutions of each siRNA were diluted 20× to 1 × 10⁻⁶m in a separate aliquot of Opti‐MEM media. The two solutions were then mixed at a 1:1 by volume for another 15 min at room temperature. Afterwards, the final mixture was added into cell culture medium without antibiotics to achieve a final concentration of 100 × 10⁻⁹m for each siRNA in 3 µg mL^–1 Lipofectamine 2000. Cells were then incubated with siRNA for 24 h at 37 °C with 5% CO₂. Cells treated with Lipofectamine 2000 alone were used as a negative control group.