Polyjet in vitro dna transfection reagent

The 3T3 protocol-immortalized wild-type p27 (p27+/+) and p27-deficient (p27−/−(Δ51)) mouse embryonic fibroblasts (MEFs) were described in previous study [50 (link)], and were cultured in DMEM with 10% FBS. Human invasive bladder cancer cell line UMUC3 and T24 [36 (link), 51 (link)] was cultured in DMEM with 10% FBS and DMEM/Ham’s F-12 (1:1) with 5% FBS, respectively, while human transitional bladder cancer HT1197 [52 (link)] was cultured in MEM with 10% FBS. Cell transfections were performed with PolyJet^TM DNA in Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD), according to the manufacturer’s instructions. For stable transfection, MEFs were subjected to selection with hygromycin B (200 μg/mL) or puromycin (2.0 μg/mL) depending on the different antibiotic resistance of the plasmids transfected. Cells surviving from the antibiotic selection were pooled as mass stable transfectants. UMUC3 and T24 transfectants were selected by puromycin (0.4 μg/mL).

For knockdown of SESN2, BECN1, and HGK, pGFP-V-RS plasmid encoding shRNA against human SESN2 (TG301755), BECN1 (TG314484), and HGK (TG320615) were purchased from OriGene (Rockville, MD). For luciferase reporter assays, the AP-1-Luc (60612) and SESN2-Luc (HPRM12429-PG02) were purchased from BPS Bioscience (San Diego, CA) and OriGene, respectively. The luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The cells were stably transfected by using PolyJet^TM DNA In Vitro Transfection Reagent (SignaGen Laboratories), according to the manufacturer’s instruction.

Protein synthesis inhibitor cycloheximide (CHX) was bought from Calbiochem (San Diego, CA, USA). The dual luciferase assay kit, TRIzol reagent and SuperScriptTM First-Strand Synthesis system were obtained from Promega (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA), respectively. PolyJet^TM DNA in Vitro Transfection Reagent was purchased from SignaGen Laboratories (Rockville, MD, USA). The plasmids of CRISPR/Cas9 RAC1 and its control vector were bought from Open Biosystems (Thermo Fisher Scientific, Pittsburgh, PA, USA). miR-145 promoter luciferase plasmid, miR-365a promoter luciferase plasmid, RAC1 3′UTR luciferase plasmid, Dicer 3’UTR luciferase plasmid, miR-365a overexpression plasmid were constructed by us. A fragment spanning 1228 bp relative to the mRNA 3’UTR site of human RAC1 genomic sequence was produced by PCR with the forward primer 5′- CCG CTC GAG CTT CGC ACT CAA TGC CAA CT -3′ and the reverse primer 5′- TCC GAG CTC GAC CCA AAG GAA CAT CAA TAG G -3′. The PCR products were subcloned into the XhoI and SacI sites of pMIR-Report vector (Promega Co., E1751) to generate the RAC1 3′UTR luciferase plasmid. The construct was confirmed by DNA sequencing (GENEWIZ). All transfectants were used as a mass pool culture rather than individual clones.

HEK-293T cells were transfected with pAAV-IRES-EGFP or pAAV-IRES-SARS-CoV-2-S-EGFP as the effector cells by PolyJet^TM DNA in vitro Transfection Reagent (SignaGen, USA). Huh 7 cells/Vero E6 cells (1 × 10⁴) expressing ACE2 receptor were incubated in 96-well plates at 37°C for 5 h followed by the addition of 293T/EGFP or 293T/SARS-CoV-2-S/EGFP cells with or without compounds. After co-culture at 37°C for 12 h, three fields in each well were randomly selected to count fused and unfused cells under an inverted fluorescence microscope (Nikon Eclipse Ti-S). The percent inhibition of cell-cell fusion was calculated using the following formula, as described elsewhere [1 − (E − N)/(P − N)] × 100%. “E” represents the percentage of cell-cell fusion in the experimental group. “P” represents the percentage of cell-cell fusion in the positive control group, where 293T/SARS-CoV-2- S/EGFP cells were used as effector cells to which no compound was added. “N” represents the percentage of cell-cell fusion in the negative control group, in which 293T/EGFP cells were used as effector cells. The IC₅₀ was calculated using CalcuSyn software. Samples were tested in triplicate, and all experiments were repeated twice.

Cell transfections were performed with PolyJet^TM DNA in Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD, USA) according to the manufacturer's instructions. For stable transfection, cell cultures were subjected to hygromycin B (200-400 μg/mL), G418 (500-1000 μg/mL) or puromycin (0.2-0.3μg/mL) and cells surviving from the antibiotics selection were pooled as stable mass transfectants.

Mitochondria Catalase expression plasmid (pZeo/mCat) and its parental control vector were kindly provided by Dr. J. Andres Melendez (Center for Immunology and Microbial Disease, Albany Medical College) as described before [31 (link), 32 (link)]. HCT116 cells were transfected with mCat and its corresponding control vector by using PolyJetTM DNA In Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD, USA) following the manufacturer's instructions and stable transfectants were selected by Zeocin-resistant selection.