Polyethylenimine transfection reagent

Lentiviral particles were made using cloned plasmid vectors, either at the Shared University Research Facility (University of Edinburgh), or in our own laboratory. Briefly, 10 µg gene-specific lentiviral vector was mixed with 7.5 µg lentiviral packaging vector psPAX2 (Addgene) and 2.5 µg envelope protein producing vector pCMV-VSV-G (Addgene) and transfected into HEK 293 T cells in a 10 cm² dish using polyethylenimine transfection reagent (Polysciences, 23966). After 48 h virus was purified by first filtering the supernatant media with a 0.45 µm filter, followed by virus concentration using Lenti-X Concentrator (Takara Bio, 631232) and resuspension of viral particles in PBS.

NIH3T3 cells were obtained from the RIKEN Cell Bank. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 37°C. Sodium ascorbate or its stabilized form, AA2G, was supplied as required at concentrations of 250 μM; however, the culture medium included a sufficient amount of ascorbate to facilitate collagen biosynthesis in many cases. Cells were seeded at a density of 1.5 × 10⁵ on 35-mm dishes and cultured for 24 h. Cells were treated with 1 μg of a plasmid mixed with 6 μl of polyethylenimine transfection reagent (Polyscience) following the manufacturer’s instructions. Medium was changed 24 h after transfection and subjected to analysis. To detect secreted collagen, cells were cultured on fibronectin-coated dishes. The HSC clones, CFSC-2G and -5H cells, were cultured in DMEM supplemented with 10% fetal bovine serum, 1× non-essential amino acids solution (FUJIFILM Wako Pure Chemical), 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 37°C. The transfection procedure was identical to that for NIH3T3 cells. Cells were stimulated with 1 ng/ml of TGF-β for 3 h as needed.

Cell transfection was used for ∆Np63α ectopic expression in WWP1 stably expressing MCF-10A cells. Human ∆Np63α was sub-cloned into pCMV-Tag2b, and transfected to MCF-10A cells using polyethylenimine transfection reagent (Polysciences, 23966). After cultured at 37 °C for 24 h, the cells were collected and used for wound-healing assay.

For plasmid transfections, cells were transfected at 70–90% confluency using Polyethylenimine transfection reagent (Polysciences) or Lipofectamine 2000 transfection reagent (Invitrogen) according to manufacturer’s instructions. Plasmids used: CDT1-GFP (cloned in pCDNA 3.1/EGFP), Geminin-dHcRed (pdiHcRed-N1), GFP (pCDNA3.1/EGFP) and dHcRed (pdiHcRed-N1) were used from Xouri et al. (2007) (link). Transfections of siRNA duplexes were performed with Lipofectamine RNAiMAX (Invitrogen) according to manufactures instructions. siRNAs used: siLuciferase: 5′ CGUACGCGGAAUACUUCGAdTdT 3′, siCDT1 #1: 5′ AACGUGGAUGAAGUACCCGACdTdT 3′, siCDT1 #2: 5′ CCUACGUCAAGCUGGAdTdT 3′ were used in final concentration of 100 nM for 48 h and were purchased from Eurofins MWG. Pre-designed siGeminin: 5′ GAAUGACCACUUAACAUCUdTdT 3′ and Nontargeting siRNA were purchased from Thermo Scientific (Ambion negative control #1) and were used in a final concentration of 20 nM for 24 h of incubation. The following reagents were used to treat cells for the indicated time at the indicated final concentration before collection: HU (Hydroxyurea, Sigma), Etoposide (Sigma), CPT (Camptothecin, Sigma).

Hepatocellular carcinoma cell lines HepG2, PLC, SK-Hep1, colorectal cancer cell line SW480 and lentivirus packaging cell HEK293T were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Invitrogen). In this study, for establishing stable cell lines with ELECTS, ELECTS and SB100X (Addgene #34879) plasmids (10:1) were co-transfected into HepG2 and SK-Hep1 cells using lipid nanoparticle Lipofectamine 3000 (Thermo Fisher #L3000015), and the cells were selected by puromycin for ~ 5 days. Transmission electron microscopy (TEM) was conducted. Zeta potential and physicochemical properties of nanomaterials were determined by dynamic light scattering (DLS) with a Malvern Zetasizer, NANO ZS (Malvern Instruments Limited, UK). For the lentiviral vector, pCDH plasmids were co-transfected into HEK293T cell with the packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using polyethylenimine transfection reagent (Polysciences, #23966). Viral particle supernatants were harvested and concentrated using 44% PEG8000 (Sigma-Aldrich, #89510) and 4 M NaCl. HepG2 and SK-Hep1 cells were infected with the lentiviruses in the presence of 5 ug/mL Polybrene (Yeasen, #40804ES76) and selected by puromycin for 5–7 days.