The human NKCC1 clone (pcDNA3.1) was provided by Dr. Shmuel Muallem at the National Institutes of Health at Bethesda, MD, United States. Plasmid DNAs were incubated in 200 μl of Jet prime buffer (Polyplus, Graffenstaden, France, #B200225) and mixed with 4 μl of the transfection reagent (Polyplus; #21Y0910L1) for 10 min. DNAs were then transferred into HEK293Tcell plates containing culture medium, and all procedures were performed according to the manufacturer’s protocol (Polyplus).
Polyplus
Manufactured by Polyplus Transfection
Sourced in France
Polyplus is a line of laboratory equipment designed for DNA/RNA transfection. It functions as a versatile tool for introducing genetic material into cells, enabling researchers to study gene expression and perform various cell-based assays.
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Lab products found in correlation
Jetprime, by Polyplus Transfection (2 mentions)
Jetprime buffer, by Polyplus Transfection (1 mentions)
Small interfering rnas sirna, by GenePharma (1 mentions)
Mirvana mirna inhibitor, by Thermo Fisher Scientific (1 mentions)
Rat igg1κ, by BioLegend (1 mentions)
Armenian hamster isotype control, by BioXCell (1 mentions)
Transfection reagent, by Roche (1 mentions)
Sh nc, by Genechem (1 mentions)
Invivofectamine 3, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Hepes buffer, by Corning (1 mentions)
Low protein binding durapore membrane, by Merck Group (1 mentions)
Colorimetric reverse transcriptase assay, by Merck Group (1 mentions)
Dh5α cells, by New England Biolabs (1 mentions)
Si con, by Thermo Fisher Scientific (1 mentions)
Interferin transfection reagent, by Polyplus Transfection (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Prl cmv reference plasmid, by Promega (1 mentions)
Renilla glo luciferase assay, by Promega (1 mentions)
Recombinant ifn β, by PBL Assay Science (1 mentions)
13 protocols using polyplus
1
Transfecting Human NKCC1 in HEK293T Cells
2
Regulation of FOXC1 by miR-149
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The sequence of pre-miR-149 was obtained from the miRbase database, and the binding sequences of FOXC1 and miR-149 were obtained from TargetScan. The miR-149 expression vector was constructed using synthetic oligonucleotides and cloned into the pcDNATM6.2-GW/EmGFP vector between the EcoRI and HindIII sites. The 3′-UTR of FOXC1 was synthesized and cloned into the pmiR-GLO vector between the SacI and XhoI sites. Small interfering RNAs (siRNAs) targeting FOXC1 and NC were purchased from GenePharma Biotech (Shanghai, China). The sequences are presented in Table 1 . Vector or siRNA transfection was performed according to standard protocols using PolyPlus (PolyPlus, Illkirch-Graffenstaden, France).
3
Knockdown of vtRNA1-1 in Cell Lines
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A constitutive vtRNA1-1 knockdown cell line was constructed analogous to the described MVP knockdown cell lines (see above). The target sequence on vtRNA1-1 was the following: 5′-GGCUGGCUUUAGCUCAGCG-3′. The whole shDNA sequence for the vtRNA1-1 knockdown is listed in Supplementary Table 2 .
To reduce endogenous vtRNA1-1 levels in HeLa cells or HS578T cells, chemically modified chimeric ASOs were used. Therefore 5 × 106 HeLa cells were transiently transfected (jetPEI, Polyplus) with 1 μM ASOs (Exiqon), directed against vtRNA1-1 or as a control against vtRNA1-2. After 24 h, cells were collected by centrifugation (450g, 5 min), stained with Annexin V and analysed by flow cytometry (as described above). ASOs were designed as RNA/DNA/RNA chimeric oligonucleotides with a phosphorothioate backbone. Ten central deoxyribonucleotides are flanked by five 2′-O-methyl modified ribonucleotides on both sides (Supplementary Table 2 ).
To reduce endogenous vtRNA1-1 levels in HeLa cells or HS578T cells, chemically modified chimeric ASOs were used. Therefore 5 × 106 HeLa cells were transiently transfected (jetPEI, Polyplus) with 1 μM ASOs (Exiqon), directed against vtRNA1-1 or as a control against vtRNA1-2. After 24 h, cells were collected by centrifugation (450g, 5 min), stained with Annexin V and analysed by flow cytometry (as described above). ASOs were designed as RNA/DNA/RNA chimeric oligonucleotides with a phosphorothioate backbone. Ten central deoxyribonucleotides are flanked by five 2′-O-methyl modified ribonucleotides on both sides (
4
Virus Transfection in 12-well Plates
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5
Immunomodulation of Melanoma Progression
6
Pseudovirus Production for HIV/Ebola Research
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HIV bearing Ebola virus and VSV GPs were generated by liposome-mediated transfection system (Polyplus; 25Y1801N5) as follows. 293T cells were seeded into 6-well plates at 7 × 105 cells per well and transfected with a total of 2 μg plasmid DNA when cells yielded a density of 60 to 80% confluence. Amounts of 0.4 μg GP or VSV-G and 1.6 μg pSG3.Δenv.cmvFluc were utilized according to the optimized results. The cell supernatants were harvested 48 h after transfection, centrifuged at 3,000 rpm for 10 min, filtered through a 0.45-μm-pore filter, and frozen in aliquots at −80°C. Titers of the pseudovirus were determined by an HIV p24 ELISA kit (Key-Bio, K12P2401).
7
OSCC Cell Line Genetic Manipulation
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Human OSCC cell lines (CAL-27 and UM-SCC-1) were used in this research, and the source and culture conditions were the same as in previous study [5 (link)]. Polyplus (Polyplus-transfection, France) was utilized to transfect Negative Control (NC), SMYD3 siRNAs (RiboBio, Guangzhou, China) and HMGA2 siRNAs (RiboBio, Guangzhou, China) into OSCC cells according to the manufacturer’s protocol, and cells were collected after 48–72 h transfection. The virus (containing short hairpin SMYD3 (shSMYD3) or shNC, Genechem, Shanghai, China) were transduced into OSCC cells with a multiplicity of infection of 100, as recommended. Roche Transfection Reagent (Roche, Switzerland) was utilized for the transfection of SMYD3, histone methyltransferase-inactive mutant-SMYD3 (EEL) [15 (link)], and HMGA2 plasmids (Weizhenbio, Jinan, China) according to the provided protocol, and cells were collected after 48 h transfection. The procedures of RNA extraction and qRT-PCR were as shown before [2 (link)]. The sequences of siRNA/shRNA and the primers used in this experiment are listed in Additional file 2 : Table S6.
8
Nucleolin Depletion Modulates Inflammation
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Ncl depletion in mouse lung- or liver-resident cells was mediated by siRNA against NCL with in vivo-jetpei (Polyplus) or Invivofectamine 3.0 (Invitrogen), respectively. According to the manufacturer’s instructions, the siRNA-in vivo-jetpei complex (120 μg siRNA) was injected twice or siRNA-Invivofectamine complex (50 μg siRNA) was injected intravenously into the tail veins of 4-week-old C57BL/6N mice. Two days later, LPS (300 μg kg−1) was intravenously injected to the tail veins of mice for 10 h, and then the level of inflammatory RNAs, proteins, or Ncl knockdown was evaluated by qPCR or ELISA. We monitored control or lung-specific Ncl-depleted mice survival every 4 h for 2 days after intravenous administration of PBS or LPS (6 mg kg−1).
9
Production of SARS-CoV-2 Spike Pseudotyped Lentivirus
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SARS-CoV-2 Spike pseudotyped lentivirus was produced as previously described (Crawford et al., 2020 (link)). Briefly, HEK293T cells were transfected with 1 μg pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI), a lentiviral backbone plasmid expressing luciferase under a CMV promoter and an IRES followed by ZsGreen, 0.22 μg HDM-Hgpm2 (BEI), a lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter, 0.22 μg HDM-tat1b (BEI), a lentiviral helper plasmid expressing HIV Tat under a CMV promoter, 0.22 μg pRC-CMV-Rev1b (BEI), a lentiviral helper plasmid expressing HIV Rev under a CMV promoter, and 0.34 μg of the plasmid encoding HDM-SARS2-Spike-delta21 using polyethylenimine (Polyplus) in serum-free Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) supplemented with 25 mM HEPES buffer (Corning). Media was changed to D10 24h post-transfection. After 48h, pseudotyped lentivirus was harvested by filtering supernatant through a 0.45 μm low protein binding durapore membrane (Millipore). Frozen aliquots were stored at −80°C and viral concentrations were quantified using the colorimetric Reverse Transcriptase Assay (Sigma-Aldrich). All packaging plasmids were propagated in DH5α cells (NEB).
10
Pseudovirus Production for HIV/Ebola Research
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HIV bearing Ebola virus and VSV GPs were generated by liposome-mediated transfection system (Polyplus; 25Y1801N5) as follows. 293T cells were seeded into 6-well plates at 7 × 105 cells per well and transfected with a total of 2 μg plasmid DNA when cells yielded a density of 60 to 80% confluence. Amounts of 0.4 μg GP or VSV-G and 1.6 μg pSG3.Δenv.cmvFluc were utilized according to the optimized results. The cell supernatants were harvested 48 h after transfection, centrifuged at 3,000 rpm for 10 min, filtered through a 0.45-μm-pore filter, and frozen in aliquots at −80°C. Titers of the pseudovirus were determined by an HIV p24 ELISA kit (Key-Bio, K12P2401).
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