Lipfectamine 2000

2C::tdTomato reporter (Addgene plasmid #40281) ESCs (Macfarlan et al., 2012 (link)) were cultured in GMEM (Life Technologies, Carlsbad, CA), 10% KnockOut Serum Replacement (ThermoFisher Scientific, Waltham, MA), 2 mM L-glutamine (Life Technologies), 0.1 mM MEM non-essential amino acids (Life Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate (Sigma), 0.05 mM 2-mercaptoethanol (Life technologies), LIF (1000 U/ml; ESGRO; Merck Milipore) with 2i (PD0325901 (1 µM; Stemgent, Cambridge, MA) and CHIR99021 (3 µM; Stemgent, Cambridge, MA). For Dppa3 over-expression, a Tet-ON3G inducible vector containing Dppa3 CDS was transfected into ESCs with Lipfectamine 2000 (ThermoFisher Scientific, Waltham, MA). 500 ng/ml of DOX was added on day 0. For Dppa3 knockdown experiment, ON-TARGETplus SMARTpool (Dharmacon, Lafayette, CO) which contain four siRNA targeting Dppa3 was transfected into ESCs using Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific, Waltham, MA). The Dppa3 siRNA SMARTpool includes the following siRNA sequences: GGAUGAUACAGACGUCCUA; UAGAUAGGAUGCACAACGA; AGAAAGUCGACCCAAUGAA; GAGUAUGUACGUUCUAAUU. ON-TARGETplus Non-targeting (scramble) siRNA (Dharmacon) was transfected as negative control. Td-tomato expression was analysed with BD LSRFORTESSA (BD Biosciences, San Jose, CA).

F9 cells were plated as described above and reverse transfected using Lipfectamine 2000 (Thermo Fisher Scientific). Briefly, 200,000 cells were seeded in 35 mm gelatin-coated plates already containing 4 μg of DNA plasmid. Culture media was replaced 6 h post transfection and cells were allowed to grow for 24 h. Following incubation, media was changed and cells were selected with 2 mg/ml G418 (Gemini Bio-Products) for 2 weeks. Cells were trypsinized and seeded at low density allowing single colony formation. Single clones were selected and propagated for downstream analysis. pcDNA 3.1 plasmid was generously provided by Dr. Robert C. Cumming and DDK-MYC-mPDK1 overexpression construct was purchased from Origene.

MiRNA-210 inhibitor, miRNA-210 inhibitor negative control (miRNA-210-NC), siRNA1-EGR3 (si1-EGR3), siRNA2-EGR3 (si2-EGR3), and siRNA negative control (si-NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). HepG2 and HepG2.2.15 cells were digested with 0.25% trypsin and seeded into 24-well plate at a density of 1.3 × 10⁵ cells/well. When reaching 90% confluence, cells were transfected with the above agents using Lipfectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells without transfection were considered as the Blank group. After 48 h of transfection, cells were used for further assays.

siRNA transfections were performed in ES cells using Lipfectamine 2000 and OptiMEM (Thermo Fisher Scientific). ES cells were plated 5-7 h before transfection at a density of 5 × 10⁵ cells per well of a 6-well plate and transfected with 100 pmol siRNA, according to the manufacturer’s standard recommendations. A SMARTpool of four independent siRNAs was used to knockdown Usp9x (Dharmacon), and a nontargeting siRNA (siGenome siControl #2, Dharmacon) was used as a control. Transfections were performed in ES-FBS medium without antibiotics, and the medium was replaced the next morning with complete ES-FBS. Cells were harvested for counting and colony formation assays or western blots 48 h after transfection.

For poly (I:C) transfection, cells were transfected at ~70% confluence with the final concentration of 50 μg/ml poly (I:C) using Lipfectamine 2000 (Invitrogen) following the manufacturer’s protocol. For gene knockdown, 150 nmol/L of double-stranded RIP1 siRNA oligonucleotides 5′-UGCUCUUCAUUAUUCAGUUUGCUCCAC-3′, RIP3 siRNA oligonucleotides 5′-UAACUUGACGCACGACAUCAGGCUG-3′, or scrambled siRNA oligonucleotides 5′-UUCUCCGAACGUGUCACGU-3′26 (link) were chemically synthesized (Beijing AuGCT DNA-SYN Biotechnology, China), and cathepsin D siRNA was purchased from Santa Cruz Biotechnology. The siRNA was transfected using Lipfectamine 2000. Quantitative PCR was performed to identify the knockdown efficacy using the following primers: RIP1, 5′-TGGGAAAGCACTGGAAAAAC-3′ and 5′-GTCGATCCTGGAACACTGGT-3′; RIP3, 5′-TTTGGCCTGTCCACATTTCAG-3′ and 5′-GGTTGGCAACTCAACTTCTCTT-3′; Cathepsin D, 5′-TGCTCAAGAACTACATGGACGC-3′ and 5′-CGAAGACGACTGTGAAGCACT-3′. At 36 hours post-transfection, cells were infected with ADV (MOI = 0.1) in the presence or absence of dsCARE. The antiviral efficacy of dsCARE was examined by fluorescent microscopy.

The shuttle plasmids pDC315, pDC315-E1A, pDC315-mE1A and pDC315-EGFP were co-transfected into HEK-293 cells with adenoviral cytoskeleton plasmid pBHGE3 (Microbix Biosystems Inc.) using Lipfectamine 2000(Invitrogen) according to manufacturer's instructions respectively. The plaque showed at about 9-14 days after transfection. The transgenes were identified by PCR using specific primers for E1A (mE1A) or EGFP. The adenoviruses Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A and Ad-DC315-EGFP (Schematic diagrams were shown in Figure 1B) were grown in HEK293 cells to amplify and titrate by tissue culture infection dose 50 (TCID50) methods. Virus stocks were stored in adenoviral buffer (Ad buffer) (10 mM Tris-HCl, pH 8.0; 2 mM MgCl₂; 4% sucrose).