Ambion silencer select pre designed sirna

Calcium- and magnesium-free phosphate-buffered saline was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). PI was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Poly(I:C)-HMW/LyoVec™ (Poly(I:C)), which is a complex of the synthetic double-stranded RNA analog poly(I:C) and a transfection reagent (LyoVec™), were purchased from InvivoGen (San Diego, CA, USA). Annexin V-FITC was purchased from BioLegend, Inc. (San Diego, CA, USA). Anti-rabbit horseradish peroxidase (HRP)-conjugated IgG and anti-mouse HRP-conjugated IgG secondary antibodies, anti-Mfn1 (cat. no. 14739), anti-OPA1 (cat. no. 80471), anti-Drp1 (cat. no. 5391), anti-eIF-2α (cat. no. 9722), anti-phospho-eIF-2α (cat. no. 3597), anti-β-actin (cat. no. 4967) monoclonal antibodies, and SignalSilence^® Mfn1 (cat. no. 13303) siRNA were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-DAP3 (cat. no. 610662) monoclonal primary antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Ambion Silencer^® Select Pre-designed siRNA against the gene-encoding Drp1 (cat. no. s19560), the gene-encoding DAP3 (cat. no. s1506), and Silencer^® Select Negative #1 Control (cat. no. AM4611) siRNAs were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

WT or Rtn4-KO U2OS cells were plated into 35-mm imaging dishes (250,000 cells/dish). After 20 h, the WT cells were transfected with 80 nM Ambion Silencer Select negative control 1 siRNA (4390843), and the Rtn4KO cells were treated with 40 nM RTN1 Ambion Silencer Select Pre-designed siRNA (s12378) and 40 nM RTN3 Ambion Silencer Select Pre-designed siRNA (s20162) according to the DharmaFECT protocol. After 24 h, medium was replaced, and cells were transfected with 2 µg SNAP-KDEL using Lipofectamine 2000 according to the manufacturer’s protocol. Medium was replaced after 6 h, and cells were imaged 24 h later.

PBS(−) (Ca²⁺, Mg²⁺-free Dulbecco’s phosphate buffered saline) and mouse anti-α-tubulin antibody (cat. no. 017-25031) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V Binding Buffer, FITC-labeled annexin V (FITC-annexin V), and rabbit anti-p63(ΔN) antibody (#619001) were purchased from BioLegend (San Diego, CA, USA). Rabbit anti-cleaved caspase-3 antibody (#9661), rabbit anti-β-actin antibody (#4967), horse-radish peroxidase (HRP)-labeled anti-rabbit IgG antibody (#7074), and HRP-labeled anti-mouse IgG antibody (#7076) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Rabbit anti-importin alpha 5 (a.k.a. KPNA1) polyclonal antibody (18137-1-AP) was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Mouse anti-LaminB1 antibody (sc-365962) was purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Ambion Silencer^® Select Predesigned siRNA against the gene encoding ΔNp63 (#1: no. s16413, #2: no. s16411), KPNA1 (no. s223980), and Silencer^® Select Negative #1 Control siRNA (no. 4390843) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).