siRNA Knock-Down in Thp-1 Cells

For siRNA knock-down of human TIFA transcript in Thp-1 cells, we used the Flexitube Qiagen siRNA Assays, containing four different validated siRNA variants against human TIFA (Flexitube GeneSolution: Hs_TIFA-6, Hs_TIFA-7, Hs_TIFA-8, Hs_TIFA-9, each at 10 µM stock concentration), according to the manufacturer’s instructions. Thp-1 cells (2 × 10⁵ per well) were transfected with siRNAs using the LONZA nucleofector kit SG (nucleofector 4D), with 1.25 µM of siRNA Mix for each siRNA per well (5 µM total of siRNA per well if all were combined, in strip well transfection cuvettes). Immediately after nucleofection, all cells from each cuvette well were resuspended in 1 ml of fresh medium (RPMI1640, 10% FCS) per well, each seeded in 24 well plates, and let acclimatize for 72 h. For each experiment, transfection mix for mock transfection (transfection agent without RNA) and Allstars Negative Control siRNAs Mix (Qiagen) were transfected in separate wells alongside, as negative controls for the knock-down. After 72 h, the cells were again incubated in fresh medium and subsequently incubated in the presence of pure ADP-heptose (5 µM per well), as indicated in the figure captions. At the end of the co-incubation period, cells were gently scraped off the wells, collected including their supernatants, which were then harvested by centrifugation for cytokine ELISA. Cells recovered in the pellets by centrifugation from the supernatants were immediately resuspended in RNA-Later (Agilent) for undamaged RNA isolation. RNA was subsequently isolated from each cell pellet as described above, quality-tested, and used to verify the specific knock-down by qRT-PCR for TIFA transcript (normalized in each condition to Hs_GAPDH transcript). For luciferase detection after siRNA transfection, Thp1_luc cells (2 × 10⁵), containing the NF-κB luciferase reporter, were transfected with siRNA or mock as described above in one strip well, then each transfection well was distributed further into 5 wells of a 96-well plate, in 100 µl of medium (final cell count per well of ca. 4 × 10⁴). The cells were again kept for 72 h for expressing the siRNA. Subsequently, medium was changed to 50 µl fresh RPMI, the cells were co-incubated with pure ADP-heptose for 4 h and then subjected to lysis and luciferase measurement (Promega Steady-Glo firefly luciferase substrate).