On targetplus nontargeting control pool

For experiments involving use of siRNA, 5–12 × 10⁶ CD25⁻CD4⁺ T cells from individual donors were resuspended in 100 µl of Nucleofection buffer solution for human primary T cells (Nucleofector Kits for Human T Cells, Lonza) containing 2 µM of ON‐TARGETplus PPP1R11 siRNA pools or ON‐TARGETplus nontargeting control pool (both Dharmacon, GE Healthcare). To deconvolute the effect of the siRNA pool, confirmation experiments were also performed with 2 individual PPP1R11 siRNAs (contained in the pool of 4 siRNAs above), which are denoted as PPP1R11‐07 and PPP1R11‐08. The cells were transfected using program U‐014 of the Amaxa Nucleofector™ 2b device using the manufacturer's recommendations. Following nucleofection, the cells were transferred to prewarmed X‐VIVO 15 medium and incubated for 4.5 days at 5% CO₂/37°C unless otherwise mentioned. The medium was changed once following 5 h of incubation. The cells were either directly frozen for studying the unstimulated state or stimulated with particular stimulations for specific time periods depending on the nature of further analyses.

U937 cells were transfected with CCR1 or PLA2G6 human siRNA ON-TARGETplus SMARTpool (GE Dharmacon, Lafayette, CO, USA), at a concentration of 30 pmol siRNA per 1 × 10⁶ cells, using the Amaxa® Cell Line Nucleofector® Kit C from Lonza (Cologne, Germany). ON-TARGETplus Nontargeting Control Pool (GE, Dharmacon) was included as a negative control. The cells were incubated under standard conditions for 48 hours before IL-1β release studies were performed. The efficiency of gene silencing was analyzed by real-time RT-PCR (CCR1 and PLA2G6) and Western blotting (iPLA2). The protein expression of CCR1 by U937 cells was below the threshold of detection in both Western blotting and flow cytometry.

siRNAs for human IRF1 (3659, ON-TARGETplus smart pool including 4 target sequences: GGGCUCAUCUGGAUUAAUA, UGAACUCCCUGCCAGAUAU, GCUCAGCUGUGCGAGUGUA, GAAGGGAAAUUACCUGAGG), siRNA for human IRF3 (3661, ON-TARGETplus smart pool including 4 target sequences: CGAGGCCACUGGUGCAUAU, CCAGACACCUCUCCGGACA, GGAGUGAUGAGCUACGUGA, AGACAUUCUGGAUGAGUUA) and ON-TARGETplus non-targeting control pool were purchased from GE health, Dharmacon. 3 x 10⁴ A549 cells were transfected with 25 μM of different siRNAs with Lipofectamine RNAiMAX complexes (Invitrogen) according to the manufacture’s instruction (reverse transfection). After 16 h of incubation, media was replaced by complete cell culture media without antibiotics. After 40 h of transfection, cells were infected with RSV-HD at a moi of 1.5 TCID₅₀/cell for 10 h. Cells were harvest with either TRIzol for RNA or NP-40 lysis buffer for protein analysis. As control, cells were treated only with Lipofectamine RNAiMAX transfection reagent.