Silencer select validated sirna

PBECs cultured in 6-well plates (60%–80% confluence) were transiently transfected with 10 nM IFN regulatory factor 3 (IRF3) siRNA (Silencer^® Select Validated siRNA, s7507; Ambion, Life Technologies, Carlsbad, CA, USA), 10 nM PIK3CD siRNA (Silencer^® Select Validated siRNA, s10529; Ambion, Life Technologies) or 10 nM negative control (NC) siRNA (Silencer^® Select Negative Control siRNA, 4390843; Ambion, Life Technologies) using Lipofectamine^® RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. Lipofectamine^® RNAiMAX Reagent or siRNA were diluted in BEGM without hydrocortisone and antibiotics. Cells were used for experiments at 48 h after transfection.

MAC2A and TLBR-2 cells (1×10^6) were transfected with 30 nM siRNA concentration for single KD. SiRNA transfections were performed using the Cell Line Nucleofector Kit SF and Amaxa 4D Nucleofector (program DS-130 for TLBR-2, FI115 for MAC2A). Twenty-four hours after transfection, cells were harvested and plated 2.5 × 105 cells/ml. For siRNA scramble, we used a Silencer Select negative control (Ambion, Life Technologies). For PAK2 and RHOA we used a Silencer Select Validated siRNAs, ID:s10022 and ID:s759, respectively (Ambion, Life Technologies). For RHOU we used two different Silencer Selected Pre-designed siRNAs ID:224502, ID: s33826 (Ambion, Life Technologies). For YY1, we used a Silencer Select Validated siRNAs: ID:s14958 (Ambion, Life Technologies)

siRNAs against human ACSL1 (ON-TARGET plus™ SMARTpool^®, #L-011654-00, GE Healthcare Dharmacon, Lafayette, CO), human ACSL3 (custom-designed siRNA, Sense: 5′-UGUUUAUUCUGGCCUAUAAUU-3′ and Antisense: 5′- UUAUAGGCCAGAAUAAACAUU-3′, Dharmacon), human ACSL4 (Silencer^® Select Validated siRNA, #4390824, ThermoFisher Scientific, Foster City, CA), human ACSL5 (ON-TARGET plus™ SMARTpool^®, #L-006327-01, Dharmacon) and human ACSL6 (ON-TARGET plus™ SMARTpool^®, L-007748-00, Dharmacon) were independently used to transfect HeLa cells using Lipofectamine^® 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 12 pmol siRNA was diluted in 200 μl Opti-MEM^® I Medium without serum (Invitrogen) in a 12-well plate. Two μl Lipofectamine^® 2000 was added to each well containing the diluted siRNA, mixed gently and incubated for 20 min at RT. 100,000 HeLa cells (in suspension after trypsinization) were added to each well, gently mixed and incubated for 48 h at 37°C in 5% CO₂. Cells were either infected with Ct for 24 h or left uninfected. Cells were processed for confocal microscopy and for WB as described above.