Silentfect lipid reagent

siRNAs for HCRP-1, AKT, and EGFR were obtained from Integrated Biotech Solutions (Shanghai, China). The sequences of siRNAs are as follows: HCRP-1, 5′-GACACUGUUUCUUCUUCAACA-3′ AKT, 5′-GAAGGAAGUCAUCGUGGCCTT-3′ EGFR, and 5′-CACAGUGGAGCGAAUUCCUTT-3′. Transfection with siRNA was performed using siLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The pcDNA3.1-control and pcPNA3.1-BIM expression plasmids were obtained from Integrated Biotech Solutions (Shanghai, China). Transfection of the pcPNA3.1-control and pcPNA3.1-BIM plasmids into the colon cancer cells was carried out using Lipofectamine 2000 transfection reagent (Invitrogen, Shanghai, China) following the manufacturer’s protocol.
SW620 cell lines were infected with lentivirus-packing HCRP-1 shRNA vector and control vector, respectively (IBS, Shanghai, China). Target cells were established by infecting with lentivirus about 2 days and then screened out with puromycin (Sigma, Shanghai, China) for 2 weeks.

S2 cells were transfected with expression vectors by HilyMax transfection reagent (Dojindo, Tokyo, Japan) or siLentfect^™ lipid reagent (Bio-Rad Laboratories, Hercules, CA, USA).

Transfection of pcDNA3.1-control and pcDNA3.1-Rap2a expression the plasmids were carried out using Lipofectamine 2000 transfection reagent (Invitrogen, Shanghai, China) following the manufacturer’s protocol. Rap2a siRNA was purchased from Gene-Pharma (Shanghai, China) and transfected using siLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. After transfection, cells were harvested for subsequent experiments. The Rap2a overexpression 786-O cell lines and control 786-O cell lines were established by infecting with lentivirus packing Rap2a expression vector (GFP is not fused with Rap2a in this vector) and control vector respectively. Target cells were infected with lentivirus for 48 hours then selected with puromycin (Santa Cruz) for 3 weeks.

Transfection with siRNA was performed using siLentFect Lipid Reagent (Bio-Rad) for RNAi according to the manufacturer’s protocol. Gene knockdown was evaluated by reverse transcription quantitative PCR (RT-qPCR). MyD88, UNC93B1, TLR4, TLR7, TLR8, NOD1 and STING pooled ON-TARGETplus human siRNAs (Dharmacon/Thermo Scientific) were used to target MyD88, UNC93B1, TLR4, TLR7, TLR8, NOD1 and STING. MDMs were treated with 20 nM siRNA two times (day -4 and day -2) before changing to fresh medium (RPMI 1640/10% human serum), rest for 1–2 hours and challenge with TLR ligands or Mav.