Ambion silencer sirna

For gene silencing, three unique Ambion Silencer siRNAs (Thermo Fisher Scientific) targeting 13 host factors identified by AP-MS were pooled and transfected into THP-1 macrophages at a final concentration of 25 nM. To simultaneously knock down G3BP1 and G3BP2 as positive control, two unique Ambion Silencer siRNAs respectively targeting G3BP1 and G3BP2 were pooled (25 nM) and transfected into THP-1 macrophage. The same amount of non-targeting siRNA (Thermo Fisher Scientific) was transfected into THP-1 macrophages as negative control. siRNA transfections were performed with TransIT-X2 Transfection Kit (Mirus Bio) following manufacturer’s instructions. Downstream assays were conducted 48 h after transfection.
To observe viral production in transfected macrophages, 500 ng of viral genomic RNA was transfected per well in 12-well plates through the TransIT^®-mRNA Transfection Kit (Mirus Bio) following manufacturer’s instructions.

Dox HCl and Span20 were purchased from Sigma Aldrich^®, St. Louis, MO, USA. The synthesis of plier-like cationic lipid B (PCL-B) was kindly assisted by Ramkhamhaeng University and Mahasarakham University, Thailand. Cholesterol (Chol) was acquired from Carlo Erba Reagent (Milan, Italy). siAF488 was obtained from Qiagen (Santa Clarita, CA, USA). siRNA targeting apoptosis-related proteins including Mcl-1, Bcl-2, and survivin was obtained from Ambion™ Silencer™ siRNA, Thermo Fisher Scientific (Waltham, MA, USA). Lipo2k and siNT were purchased from Invitrogen (Carlsbad, CA, USA).

Cholesterol-based cationic lipids were synthesized and characterized according to a previous study [49 ] and kindly provided by Dr Boon-ek Yingyongnarongkul. The cholesterol-based cationic lipids used in this study were coded as 4as1, 6as1, and 8as1. Their structures are provided in Figure 1. Span20 was purchased from Sigma Aldrich^® (St. Louis, MO, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was purchased from Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). The control scramble siRNA (siNT) and siRNA targeting apoptosis-related proteins Mcl-1 (siMcl1) was obtained from Ambion™ Silencer™ siRNA (Cat# 4390824); (Thermo Fisher Scientific, Waltham, MA, USA). The Alexa Fluor 488-labeled siRNA (siAF488) was obtained from Qiagen (Santa Clarita, CA, USA). Annexin V-APC and SYTOX™ Green were purchased from Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). All cell-culture reagents and media were purchased from Gibco BRL (Rockville, MD, USA). The BT474 and MCF-7 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin according to the recommended protocol.