Three Silencer Select siRNA oligos (Thermo Fisher, siRNA ID: s14961, s14962, s14963) were used for each gene. Transfections were performed on day 6 of differentiation using Endo-Porter (Gene Tools). Analysis was performed 48 h after transfection. The sequences of siRNAs are listed in Table 2 .
Silencer select sirna oligos
Manufactured by Thermo Fisher Scientific
Silencer Select siRNA oligos are a set of short, double-stranded RNA molecules designed to target and silence specific genes within cells. The core function of these oligos is to induce post-transcriptional gene silencing by binding to complementary mRNA sequences and preventing their translation or promoting their degradation.
Automatically generated - may contain errors
6 protocols using silencer select sirna oligos
1
Silencing Gene Expression in Differentiation
2
siRNA-mediated Knockdown of PTGER2 and PTGER4
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Gene knockdowns were performed with Silencer Select siRNA oligos (Thermo Fisher Scientific) specific for PTGER2 (s11449) or PTGER4 (s60395), or combinations thereof, using the NEON transfection method (Thermo Fisher Scientific) as previously described55 (link). Briefly, hBMSCs (1 × 105 cells) were transfected with up to 20 nM of siRNAs or negative control siRNA (Negative Control-1), and seeded in cell culture plates with fresh growth medium (without antibiotics) for 24 h at 37 °C, 5% CO2. Medium was then replaced with osteogenic induction medium, and knockdown efficiency confirmed at selected time points by RT-qPCR.
3
Silencing NINJ1 in Human Macrophages
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All Silencer Select siRNA oligos were purchased from Ambion (Life Technologies). Individual siRNA targeting NINJ1 (ID# s9556) was used. The two Silencer Select negative control siRNAs (Silencer Select Negative Control No. 1 siRNA and Silencer Select Negative Control No. 2 siRNA) were used as a control. Three days prior to the infection, 30 nM of siRNA was transfected into the human macrophages using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Then, 24 hours after transfection, the media was replaced with fresh media containing antibiotics. Finally, 16 hours before infection, the media was replaced with fresh antibiotic-free media containing 100 ng/ml Pam3CSK4.
4
Silencing CASP4 and CASP5 in Macrophages
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All Silencer Select siRNA oligos were purchased from Ambion (Life Technologies). For CASP4, siRNA ID# s2412 was used. For CASP5, siRNA ID# s2417 was used. The two Silencer Select negative control siRNAs (Silencer Select Negative Control No. 1 siRNA and Silencer Select Negative Control No. 2 siRNA) were used as a control. Two days before infection, 30 nM of siRNA was transfected into macrophages using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. 16 hours before infection, the media was replaced with fresh antibiotic-free media containing 100 ng/ml Pam3CSK4. In parallel, siRNA-transfected cells were also transfected with 2 μg/ml of E. coli LPS strain W3110 (kindly provided by Robert Ernst) using FuGENE HD transfection reagent (Promega) for 6 hours.
5
Silencing Irg1 in Bone Marrow-Derived Macrophages
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Silencer Select siRNA oligos were purchased from Life Technologies. Irg1 siRNA s68386 and negative control siRNAs (Negative Control No. 1 siRNA 4390843 and Negative Control No. 2 siRNA 4390846) were utilized at 12 pmol per well. Transfection into BMDMs was performed using Lipofectamine RNAiMax transfection reagent (Invitrogen), following a modified protocol of the “Forward Transfection” protocol from the manufacturer’s manual. Transfection with appropriate siRNAs was performed one day after BMDM replating into 24-well tissue culture-treated plates. 8 hours after transfection, fresh media supplemented with rTNF was added to each well. After 16 hours, cells were then infected as described in the Infection section. 72 hours after the initial siRNA transfection, cells were transfected with siRNAs a second time to increase the duration of knockdown. Knockdown efficiency was confirm using qRT-PCR analysis, described below.
6
LPS and Pam3CSK4 Stimulation of Macrophages
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Recombinant proteins (PA, LFn-FlaA 310-475 , LFn-PrgJ, and LFn-YscF) were kindly provided by Russell Vance [18] . PA and LFn doses for in vitro delivery were: 1 μg/ml PA for FlaTox; 4 μg/ml PA for PrgJTox and YscFTox; 500 ng/ml LFn-FlaA 310-475 ; 8 ng/ml LFn-PrgJ; and 200 ng/mL LFn-YscF. siRNA-mediated knockdown of genes All Silencer Select siRNA oligos were purchased from Ambion (Life Technologies). For CASP4, siRNA ID# s2412 was used. For CASP5, siRNA ID# s2417 was used. The two Silencer Select negative control siRNAs (Silencer Select Negative Control No. 1 siRNA and Silencer Select Negative Control No. 2 siRNA) were used as a control. Two days before infection, 30 nM of siRNA was transfected into macrophages using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) following the manufacturer's protocol. 16 hours before infection, the media was replaced with fresh antibiotic-free media containing 100 ng/ml Pam3CSK4. In parallel, siRNAtransfected cells were also transfected with 2 μg/ml of E. coli LPS strain W3110 (kindly provided by Robert Ernst) using FuGENE HD transfection reagent (Promega) for 6 hours.
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