Non targeting sirna

UNC5B or AR siRNA was transfected using lipofectamine transfection reagent (Invitrogen, Tokyo, Japan), according to the manufacturer’s instructions. UNC5B siRNA (UNC5B si#1: 5′-UACGUGUUCACGGGCGAGUCCUAUU-3′, 5′-AAUAGGACUCGCCCGUGAACACGUA-3′, UNC5B si#2: 5′-UCCACAGAGCUCACCUGCAAGAUCU-3′, 5′-AGAUCUUGCAGGUGAGCUCUGUGGA-3′, UNC5B si#3: 5′-GAGGGCCAGAUAUUCCAGCUGCAUA-3′, 5′-UAUGCAGCUGGAAUAUCUGGCCCUC-3′) (HSS137260, 137261, 137262, stealth, Thermo Fisher Scientific, USA) and AR siRNA (AR si#1: 5′-GACUCCUUUGCAGCCUUGCUCUCUA-3′, 5′-UAGAGAGCAAGGCUGCAAAGGAGUC-3′, AR si#2: 5′-GAUGAAGCUUCUGGGUGUCACUAUG-3′, 5′-CAUAGUGACACCCAGAAGCUUCAUC-3′, AR si#3: 5′-CCGGAAGCUGAAGAAACUUGGUAAU-3′, 5′-AUUACCAAGUUUCUUCAGCUUCCGG-3′) (HSS100619, 179972, 179973, stealth, Thermo Fisher Scientific, USA). Non-targeting siRNA (Invitrogen) was used as control.

Cells were seeded into a well of 6-well plate and were cultured to approximately 60% confluency. Subsequently,the cells were transfected with EZH2 siRNA oligonucleotides and non-targeting siRNA (Guangzhou, China) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells in the exponential growth phase were then incubated with phosphate-buffered saline (PBS) (NC group) or various concentrations of gefitinib (ZD1839), GSK343, and DZNep HCl (Selleck Chemicals LCC, USA) for 48 h.The cells were then harvested for downstream experiments.

SiRNA oligonucleotides, Silencer Select Pre‐designed siRNAs (Life Technologies, Carlsbad, CA, USA), were used for the transient silencing of SQR. For SQR, the applied sequence contained the 5′‐3′ sense, GCUCAGUAAACAUCCCGUUtt and antisense 3′‐5′, AACGGGAUGUUUACUGAGCca. This SQR‐silencing siRNA (Ambion s81773) was complementary with the mRNAs belonging to RefSeq NM_001162503.1 and NM_021507.5 genes and also targeted exon 3. For negative control, non‐targeting siRNAs were applied with the same chemical modifications for enhanced efficacy as in other Silencer Select siRNAs (Ambion). Silencing was conducted as described previously (Modis K, Faseb 2012). Cells (80,000 cells/well) were seeded into 6‐well tissue culture plates and cultured in normal culture medium to reach 50% confluence. At this point, the growth medium was replaced with Opti‐MEM medium lacking FBS and antibiotics/antimicotics, followed by transfection with 25 pmol siRNA fragments per well at 30 nM forming complexes with 7.5 ml of Lipofectamine^® RNAiMAX (Life Technologies). Control cells were transfected in parallel with non‐targeting siRNA (Life Technologies).

Indicated cells were transiently-transfected with NEDD4-targeting small interfering RNA (siRNA), sequence with 5′-UUCAAUUGCCAUCUGAAGUUUAUCC-3′ (Life Technologies, Shanghai, China). Non-targeting siRNA (Life Technologies, Shanghai, China) was used as control [26 (link)]. 5 × 10⁵ cells were seeded in 6-well plates and grown to 60-80% confluence. Transient transfection with NEDD4 siRNA into PANC-1 cells using Lipofectamine 2000 (Life Technologies, Shanghai, China) for 72 hours. Simultaneously, non-targeting siRNA was transiently transfected at the same time. In all cases, successful silencing was verified by western blot (Figure 1B, and data not shown).