Human monocytes nucleofector kit

HDAC inhibitor Tricostatin A (TSA) (Cayman) was used in cell culture at 50μM for 16hr before subsequent assays. ON-TARGETplus siRNA for knocking down gene expression of ACAT1, ACLY, CRAT, CACT or non-targeting control siRNA was purchased from Horizon Discovery. siRNA against human genes or control siRNA were incubated with a mixture of nucleofection solution (Human Monocytes Nucleofector kit; Lonza) and primary human monocytes and placed in nucleofection cuvettes subjected to program Y-010 for the Nucleofector 2b Device (Lonza). 500μL RPMI medium was immediately added into cuvettes after nucleofections. Cells were then plated in 12-well plate and incubated at 37°C under 5% CO₂ for 48hr before harvesting for the assays. Plasmid transfections using empty vector (pcDNA3.1) or human ACAT1-expression constructs were conducted with the same protocol as siRNA transfection.

NR (ChromaDex) cell incubation (0.5 mM) for 16 hours was used prior to the subsequent assays. PARP inhibitor rucaparib camsylate (Sigma-Aldrich) and CD38 inhibitor apigenin (MilliporeSigma) were used in cell culture at 10 μM or 20 μM for 8 hours, respectively. Autophagy-related inhibitors or activator were used in the following concentrations for overnight incubation with the cells: 3-MA (5 mM), nocodazole (1 μg/mL), chloroquine (10 μM), and verapamil (50 μM). Primary human monocytes were incubated with purine metabolism–related chemicals at the following concentrations overnight before LPS stimulation: inosine (0.5–5 μM), hypoxanthine (5 μM), uric acid (5 μM), adenine (5 μM), and methotrexate (50–100 μM). ON-TARGETplus siRNA for gene knockdown was utilized targeting CD38, sirtuins (Sirt1, Sirt2, Sirt3, Sirt5, and Sirt7), and autophagy proteins (ULK1, ATG3, ATG5) (Dharmacon, Horizon Discovery). siRNA was incubated with a mixture of nucleofection solution (Human Monocytes Nucleofector kit; Lonza) and primary human monocytes and placed in nucleofection cuvettes subjected to program Y-010 for the Nucleofector 2b device (Lonza). After nucleofections, 500 μL RPMI medium was immediately added into cuvettes. Cells were then plated in a 96-well plate or 12-well plate and incubated at 37°C under 5% CO₂ for 36 hours before assays.