Stealth rnai pre designed sirnas

Cell culture and differentiation of P19 cells were performed as described (11 (link), 12 (link)). P19-derived neural cells were treated with 4 μM of AG1478 for day 6 and then half of the concentration for days 7 to 9. HeLa and COS7 cells were cultured in 10% FBS-containing DMEM. MDA-MB-231 cells were cultured in 10% FBS-containing RPMI1640 medium. Transfections were performed using Lipofectamine-Plus reagent, Lipofectamine 2000, or Lipofectamine RNAiMax (Invitrogen), as suggested in the instructions from the manufacturer. Immunoblotting was performed as described previously (11 (link), 12 (link)). In the protein ubiquitination experiment, cells were treated with CQ (20 μM) overnight after transfection to prevent protein degradation. Immunoprecipitation was performed using antibodies and Protein G agarose (Invitrogen), following the instructions from the manufacturer, in the presence of N-ethylmaleimide (10 mM), to prevent protein deubiquitination during the process. Immunoblot images were scanned, and densitometries were calculated using ImageJ (NIH). Establishing stable cell lines was performed as previously described (11 (link), 12 (link)). To knock down ACK or ATG5 expression, Stealth RNAi predesigned siRNAs (Invitrogen) were used (ACK #1, MSS225066; ACK #2, MSS255067; ACK #3, MSS284822; ATG5 #1, MSS247019; ATG5 #2, MSS247020; ATG5 #3, MSS247021).

All siRNAs were purchased from Invitrogen (Tokyo, Japan). Knockdown of TLR3 and TRIF was performed using sets of three specific siRNA duplexes (TLR3: #1: HSS110815; #2: HSS110816; #3: HSS110817, TRIF: #1: HSS152364; #2: HSS152365; #3: HSS175528) (Stealth RNAi™ Pre-Designed siRNAs; Invitrogen). Table 2 shows the siRNA sequences. Stealth RNAi™ Negative Control Duplexes (#12935–300, Invitrogen) served as negative control. Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) was used for reverse transfection according to the manufacturer’s instructions. The cells were transfected with a final concentration of 20 nM of each siRNA duplex set. The knockdown efficacy was confirmed by qRT-PCR and Western blotting at 72 h after transfection, and the cells were used in experiments as “TLR3 or TRIF-knockdown” cells.

A gene-knockdown experiment was performed by transfection of Stealth RNAi Pre-Designed siRNAs (Thermo Fisher Scientific) for HDAC9 mRNA (si-HDAC9) and a negative control (si-Control). Briefly, siRNA and Lipofectamine3000 (Thermo Fisher Scientific) were dissolved in Opti-MEM^® I Reduced Serum Media (Thermo Fisher Scientific) separately, and then mixed to form transfection complex for 15 min. A final concentration of 10 pmol/mL of siRNA was used for the knockdown experiments.