Liposome lipofectamine 2000

HCC cell lines, Hep3B and HepG2, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (HyClone, Logan, UT) medium supplemented with 10% fetal bovine serum (FBS, GibcoBRL; Grand Island, NY, USA) at 37 °C with 5% CO₂. MDA19 (Cat# HY-15451, MedChemExpress, USA) was dissolved in DMSO and then diluted with DMEM to a specific concentration to incubate with HCC cells. siRNA targeting CB₂ and a negative control siRNA (siNC) were synthesized by Guangzhou RiboBio Co., Ltd. and transfected into HCC cells by using Lipofectamine2000 liposomes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The sequences of siRNAs were as follows:
CB₂ siRNA1: 5′-CCAGGTCAAGAAGGCCTTT-3′;
CB₂ siRNA2: 5′- GCTTGGATTCCAACCCTAT-3′;
CB₂ siRNA3: 5′-CCTGGCCAGTGTGGTCTTT-3′;
siNC: 5′-UUCUCCGAACGUGUCACGUTT-3.

Fischer Rat Thyroid (FRT) cells were donated by Prof. Tonghui Ma from Northeast Normal University (Jilin, China); the primers and cut-gel recovery kits used were purchased from Biotech Bioengineering Corporation (Shanghai, China). Lipofectamine 2000 liposomes, TRIzol reagents, reverse transcription reagents, Geneticin (G-418), puromycin, rabbit anti-rat TRPV4 polyclonal antibodies, rabbit anti-rat β-actin monoclonal antibodies, and goat anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA, USA). F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl⁻channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quantitative real-time reverse transcriptase PCR (qRT-PCR) reagents, whole protein extraction kits, and bicinchoninic acid (BCA) protein assay kits were purchased from All Style Gold Biotechnology Co. (Beijing, China).

The siRNA-iNOS and negative controls were synthesized and purchased from Invitrogen, China. The iNOS gene sequences are iNOS siRNA1: 5′-ACAACAGGAACCUACCAGCTT-3′; 5′-GCUGGUAGGUUCCUGUUGUTT-3′; iNOS siRNA2: 5′-ACACAAGGCCAAUACCGACTT-3′; 5′-GCGUGUAUUGGCCUGUGUUTT′-3′; negative control siRNA: 5′-ACCAUAGGAUCCUACACGCTT-3′; 5′-GCGGCUAGCUUCCUUGUGUTT′-3′. siRNA was used at a concentration of 30 nM using Lipofectamine 2000 liposomes (Invitrogen) as transfection agent (0.02%). After 24 h, Tca8113 cells were transfected with siRNA according to the manufacture's protocol. The transfection of each group of cells was observed under a fluorescence microscope.

MCF-7 and SK-BR-3 were placed in 6-well plates at 2 × 10⁵ per well. Twenty-four hours after placing, siRNA against PKM2 and negative miRNA (GenePharma, Shanghai) were transfected to the cells using lipofectamine 2000 liposomes (Invitrogen) according to the manufacturer’s protocol. After culturing for 48 h, transfection efficiency was quantified by Western blotting. The sequences of siRNAs were as follows: PKM2 sense 5’- CAUCUACCACUUGCAAUUATT-3’, anti-sense 5’- UAAUUGCAAGUGGUAGAUGTT-3’; negative control sense 5’-UUCUCCGAACGUGUACGUTT’, anti-sense 5’- ACGUGACACGUUCGGAGAATT-3’. Lentivirus-based short hairpin RNA (shRNA) vector and lentivirus-based cDNA targeting the PKM2 gene were constructed by GenePharma (Shanghai, China). PKM2 shRNA was generated with CATCTACCACTTGCAATTA oligonucleotide targeting exon 10 of the PKM2 transcript. Lentiviru vector shRNA was generated with CATCTACCACTTGCAATTA oligonucleotide. Cells were selected with puromycin for 14 days at 37 °C after being infected with lentiviral. The effectiveness of transfection using lentiviruses in SK-BR-3 cells was verified by Western blotting.

Transfections of Rph-capped polyA-tailed RNAs into Huh7 cells (0.5 μg RNA per 4 × 10⁴ cells) and transfections of in vitro transcribed HCV replicons (0.4 μg) together with a capped and polyA-tailed R-Luc expressing RNA used as a normalization control (0.1 μg, derived from the XbaI/XbaI deletion variant of Rp), into Huh7-Lunet cells (4 × 10⁴), were performed at 90% confluence using the liposome Lipofectamine 2000 (Invitrogen), as the manufacturer recommended, without removing liposome–RNA complexes until cell lysis. Transfections with Rph uncapped polyA-tailed RNAs into Huh7 cells and with HCV replicons into Huh7.5[CoreE1][E2p7NS2] packaging cells were performed by electroporation (10 μg of in vitro transcribed RNA was mixed with 400 μL of cell suspension containing 1 × 10⁷ cells/mL in the case of Huh7 and 1.5 × 10⁷ cells/mL in the case of Huh7.5 packaging cells), as described elsewhere [42 (link)].