Escort 4 transfection reagent

The nucleolin targeting siRNA was obtained from Qiagen (SI02654925). LRRC59 targeting siRNA was described previously [27] (link). Scrambled control siRNA was obtained from Thermo Scientific Dharmacon (CD-001810-01-20). For siRNA transfection studies, U2OSR1 cells (1x10⁵cells/ml) were seeded out and after 24 h the cells were transfected with 50 nM of the nucleolin targeting siRNA and control targeting siRNA, and 75 nM of the LRRC59 targeting siRNA using Lipofactamine RNAiMax Transfection Reagent (Invitrogen) according to the procedure given by the company. Seven hours after transfection, 10% FBS was added to the cells, and the cells were cultured for 72 h before further experiments.
Transient expression of myc-FGF1 was performed by transfecting HEK 293 cells with plasmid DNA using Escort IV Transfection Reagent (Sigma) in Minimum Essential Medium (MEM, Gibco) according to the manufacturer’s protocol. After 7 h the medium was changed for DMEM supplemented with 10 % FBS and the cells were grown for 24 h.

Transfer plasmids for recombinant baculovirus expression vector (rBEV) generation were co-transfected with flashBACGOLD™ (Oxford Expression Technologies Ltd., Oxford, UK) genomic DNA to Sf9 cells using Escort IV transfection reagent (Sigma-Aldrich, Oakville, ON, Canada) according to manufacturer’s directions. Supernatant from each transfection was harvested 4–5 days post transfection and used to infect suspension Sf9 cultures ( $\sim$ $1.5\times {10}^6$ cells/mL) at low multiplicity of infection (MOI) for 3–4 days to amplify the rBEV. Following two rounds of amplification, the rBEV infectious virus titer (IVT) was quantified using end-point dilution assay (EPDA). Briefly, Sf9 cells were seeded at a density of $\sim$ $2.0\times {10}^4$ cells/well to each well of a 96-well plate (Fisher Scientific, Whitby, ON, Canada). Separately, the rBEV was serially diluted ( ${10}^{-2}$ to ${10}^{-8}$ ) in fresh SF900 III medium and 10 $\mathsf{\mu }$ L of each dilution was added in 12 replicates to the 96-well plate containing cells. Plates were incubated for 6–7 days at 27 ${}^{\circ }\mathrm{C}$ , after which wells were scored according to visualization of green fluorescence using a fluorescence microscope or cytopathic effects. Results were converted from TCID₅₀ and reported as plaque forming units per ml (pfu/mL) [22 ].

N- or C-terminal GFP-fused BmRNase κ cDNA was cloned into the pIZ/V5 plasmid. To prevent dimerization of GFP, site-directed mutagenesis was performed to introduce GFP A206K mutation^{61 (link)}. For the C-terminal GFP-fusion construct, the first methionine in the ORF of GFP was replaced with alanine to avoid initiation of translation from this site. The resultant plasmids were transfected into BmN4 cells using Escort IV Transfection Reagent (Sigma). After culturing for 3 days, the cells were plated on a slide glass chamber (Lab-Tek) and cultured for 18 h. The cells were then incubated with 100 nM of MitoTracker Red CMXRos (Life Technologies) for 30 min at 27 °C. After DNA mounting with ProLong Gold Antifade Reagent containing DAPI (Life Technologies), images were acquired using a Nikon A1R Confocal Laser Microscope System. The fluorescence signal intensities were quantified using ImageJ software (ver. 1.48) after background subtraction with a rolling ball radius of 50 pixels.

Sf9 protein expression and purification was performed as previously described [14 (link)]. Briefly, Sf9 cells in suspension were transiently transfected with desired DNA constructs using Escort IV transfection reagent (Sigma-Aldrich) and harvested 60–72 h later. Protein was batch purified with anti-FLAG M2 Affinity gel (Sigma-Aldrich), eluted with FLAG peptide, and buffer-exchanged using Zeba Spin Desalting columns (Pierce) into the working buffer (20 mM HEPES, 0.5 mM EGTA, 5 mM MgCl₂, 2 mM dithiothreitol (DTT), pH 7.5). Protein was further diluted into this working buffer plus 0.1 mg/ml BSA for all experiments unless otherwise described. Prior to each experiment, de-salted protein was centrifuged (2.8 x 10⁵ rcf, 10 min, 4°C) and the concentration was quantified using either an absorbance at 280 nm and a extinction coefficient calculated using ProtParam (http://web.expasy.org/ProtParam) or using mCitrine (mCit) fluorescence fit to a standard curve (FluoroMax-4, Horiba Scientific). Protein batches were made fresh and experiments performed within 72 hours of Sf9 cell harvesting.

To knockdown CES2 in H1299 or A549 cells, shRNA targeting CES2 (shCES2; senses 5’-GGTCTCCAATTCTAGTTTA-3’, antisense: 5’-TAAACTAGAATTGGAGACC-3’) and its shRNA negative control (shNC) were synthesized by GenePharma (China). After culture, lung cancer cells were transferred into 6-well plates, and incubated overnight to reach 50-70% confluence. Then, cell transfection was performed with Escort IV Transfection Reagent (L3287, Sigma-Aldrich, USA) according to the manufacturer’s protocol. The cells transfected with shCES2 or shNC were harvested 48 h post transfection, and then subjected to examination of transfection efficiency using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).

Sf9 cells were maintained in suspension culture in Gibco SF900 III serum-free medium (Fisher Scientific, Whitby, ON, Canada) as described previously [32 (link)]. Sf9 cells were transfected as adherent cultures in tissue culture treated 6-well plates (VWR, Mississauga, ON, Canada) with Escort IV transfection reagent (Sigma-Aldrich, Oakville, ON, Canada) according to manufacturer’s directions. To derive the transgenic Sf9-Cas9 and Sf9-dCas9 cell lines, parental Sf9 cells were transfected with the plasmid pOpIE2-Cas9-puro or pOpIE2-dCas9-puro, respectively. Approximately 48 h post transfection (hpt), growth medium was aspirated and replaced with fresh medium containing 5 µg/mL puromycin (Sigma-Aldrich). Selective pressure was maintained for at least 2 weeks, and resistant cells were pooled and adapted back to suspension culture. The recombinant cells were maintained in suspension culture for ~10–15 additional passages in medium containing puromycin prior to further analysis.