To investigate the effects of silenced monkey Hsp70 on PEDV replication, three small interference RNAs (siRNAs) targeting the monkey Hsp70 (siRNA-Hsp70-1, 5′-GGAGGUAUCCUCUAUGGUUTT-3′; siRNA-Hsp70-2, 5′-GCAGAAAGAAACGUGCUUATT-3′; siRNA-Hsp70-3, 5′-CCUGAUGAAGCUGUUGCUUTT-3′) were synthesized. Non-specific siRNA (siRNA-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′) was used as control. Vero E6 cells grown to approximately 30%–50% confluence in 6-well plates were transfected with siRNA-Hsp70 (40 nmol/well) or siRNA-NC (40 nmol/well), respectively, using Exfect 2000 transfection reagent (Vazyme, Nanjing, China) according to the manufacturer’s instruction. Twenty-four hours post transfection; cells were washed three times with PBS, and infected with PEDV strain CV777 at 1 MOI. At 24 hpi, samples were subjected to detection of PEDV RNA and protein by qRT-PCR and immunoblotting, respectively.
Exfect 2000 transfection reagent
Manufactured by Vazyme
Sourced in China
The Exfect 2000 is a transfection reagent designed to efficiently deliver nucleic acids, such as plasmid DNA or siRNA, into a variety of cell lines. It facilitates the uptake of these biomolecules by the target cells, enabling gene expression studies, gene knockdown experiments, and other applications that require the introduction of genetic material into cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by ExCell Bio (3 mentions)
Dual luciferase reporter assay kit, by Vazyme (3 mentions)
Pegfp n1 pcbp1, by Thermo Fisher Scientific (2 mentions)
Pegfp n1, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Nrf2 sirna, by GenePharma (1 mentions)
Control sirna, by GenePharma (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Pgl4.29 luc2p cre hygro, by Promega (1 mentions)
Fast mutagenesis system kit, by Transgene (1 mentions)
Dmem f12 1 1 medium, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Dual luciferase reporter gene assay kit, by Beyotime (1 mentions)
Lipofectaminetm 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Magna riptm rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
33 protocols using exfect 2000 transfection reagent
1
Silencing Hsp70 Inhibits PEDV Replication
2
Hsp70 Impact on PEDV Replication
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To investigate the effects of Hsp70 on PEDV replication, plasmid encoding porcine Hsp70 was transfected into Vero E6 and porcine small intestinal epithelial cells (IPEC-J2). To construct the recombinant overexpression vector, porcine Hsp70 (HSPA6, reference sequence: NM_001123127) coding sequence without the stop codon was cloned from the total cDNA of porcine kidney (PK-15) cells. The Hsp70 fragment was amplified by PCR and inserted into the multiple cloning site of the pTSB plasmid (TSE02, TranSheepBio, Shanghai, China). The recombinant pTSB-Hsp70 vectors were successfully constructed, as confirmed by DNA sequencing analysis (data not shown). To determine the effects of Hsp70 on PEDV replication, Vero E6, and IPEC-J2 cells plated on 6-well plates were transient transfected with 2.5 μg/well pTSB-Hsp70 plasmid using Exfect 2000 transfection reagent according to the manufacturer’s recommendations (Vazyme, Nanjing, China). IPEC-J2 cells were purchased from Ze Ye (Shanghai, China) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplement with 10% FBS and 1% penicillin/streptomycin. At 24 h post-transfection, cells were infected with PEDV strain CV777 at a multiplicity of infection (MOI) of 1. At 48 h post inoculation (hpi), samples were subjected to detection of PEDV RNA and protein by qRT-PCR and immunoblotting, respectively.
3
Silencing Porcine TLR2 with siRNA
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Two short interfering RNA (siRNA) sequences targeting the porcine TLR2 (siRNA210 and siRNA662) and one irrelevant interference sequence (negative control, Table 1) were designed separately (based on GenBank: NM_213761.1). Sequences were synthesized by GenePharma Company. Cells were transfected with siRNA using ExFect®2000 Transfection Reagent (Vazyme, China) according to the manufacturer's instructions. RNAs were extracted from cells after 24 h, 36 h and 48 h post-transfection, the protein was collected at 48 h post-transfection.
4
HeLa Cell Transfection and Expression
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The human cervical carcinoma HeLa cells were obtained from the First Hospital of Lanzhou University and cultured in Dulbecco's modi ed Eagle's medium (DMEM, Minghai Biochem, Lanzhou, China) supplemented with 10% fetal bovine serum (Minghai Biochem, Lanzhou, China) at a culture temperature of 37℃, 5% CO 2 in incubator (Thermo, USA). DNA transfection was carried out using Exfect2000 transfection reagent (Vazyme, Nanjing, China) as a mediator according to the manufacturer's instruction. The pEGFP-N1-PCBP1 and non-targeting negative control pEGFP-N1 was purchased from Invitrogen (Invitrogen Life Technologies, CA, USA). To transfect HeLa cells with plasmid vector, cells were plated into either 60 mm dish or a 100 mm dish and allowed to adhere for 24 h. Exfect2000 transfection reagent was utilized for the transfection. After pEGFP-N1 or pEGFP-N1-PCBP1 transfection, cells were cultured for 5 h and then the medium was replaced with fresh medium supplemented with 10% fetal bovine serum. Cells were harvested 24-48 h after transfection.
5
Knockdown of Porcine TLR2 Using siRNA
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Two short interfering RNA (siRNA) sequences targeting the porcine TLR2 (siRNA210 and siRNA662) and one irrelevant interference sequence (negative control, Table 1 ) were designed separately (based on GenBank: NM_213761.1). Sequences were synthesized by GenePharma Company. Cells were transfected with siRNA using ExFect®2000 Transfection Reagent (Vazyme, China) according to the manufacturer’s instructions. RNAs were extracted from cells after 24 h, 36 h and 48 h post-transfection. Protein was collected at 48 h post-transfection.
The sequences of siRNA targeting TLR2 used in this study (Jin et al. 2020 (link))
| Name | siRNA sequence (5'–3') |
|---|---|
| siRNA 210 | F: CCAGAUCUUUGAGCUCCAUTT |
| R: AUGGAGCUCAAAGAUCUGGTT | |
| siRNA 662 | F: CCAAAGAGUCUGAGGUCAATT |
| R: UUGACCUCAGACUCUUUGGTT | |
| Negative control | F: UGACCUCAACUACAUGGUUTT |
| R: AACCAUGUAGUUGAGGUCATT |
6
Targeted Knockdown of NRF2 using siRNA
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NRF2 siRNA and control siRNA were purchased from Gene Pharma (Shanghai GenePharma Co., Ltd). The siRNA was transfected into cells according to the manufacturer’s instruction using the Exfect 2000 Transfection Reagent (T101-01, Vazyme, Nanjing, China). For transfection, the cells were seeded in 6-well culture plates and incubated with control siRNA or NRF2 siRNA at 50 nM in a serum-free OPTI-MEM medium. Replace the medium 4 h after transfection. After incubation, the transfected cells were subjected to the treatment for the follow-up experiment. The antisense and sense was designed as 5’-AAUCAAAUCCAUGUCCUGCTT-3’ and 5’-GCAGGACAUGGAUUUGAUUTT-3’, respectively.
7
Transcriptional Regulation of IGF1 by CTCF
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Luciferase assays in HEK-293T cells were used to assess transcription activation. The promoter of IGF1 (−900 bp to +137 bp) was amplified and inserted into the plasmid vector pGL4.1. HEK-293T cells (1 × 104 cells/well) were seeded in 24-well plates until 60–80% confluence, then IGF1 reporter vectors were co-transfected with recombinant pGL4.1 luciferase reporter plasmid and wildtype or variant CTCF expression vectors with the DNA ratio 1:1 (2.5 μg each) by Exfect2000 Transfection Reagent (Vazyme). Luciferase was evaluated at 24, 36, 48, 60, and 72 h after transfection using the luciferase assay system (Promega).
8
Cell Culture and Transfection Protocols
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Cell culture media were prepared and maintained according to standard cell culture procedures. The HEK-293T (ATCC® CRL-11268) were cultured in high-glucose DMEM (Hyclone®), containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO2. B958-EBV, LCLs, and immortalized normal human liver cell line LO2 cells were cultured with RPMI1640 medium with 10% fetal bovine serum at 37°C with 5% CO2. HEK-293T cells were cultured in 24-well plates. For transient transfections, 70% confluent HEK-293T cells were transfected with 500 ng plasmid DNA per well by Hieff Trans™ Liposomal Transfection Reagent (Cat#40802, Yeasen, Shanghai, China). All cell lines were routinely tested for mycoplasma contamination.
For transient transfection, HEK-293T and LO2 cells were seeded in a 6-well plate until 60–80% confluence. The volume/mass ratio of Exfect2000 Transfection Reagent (Vazyme) to DNA was 3:1. Transfection efficiency was evaluated using quantitative real-time polymerase chain reaction (qPCR) and Western blot. After transfection for 48 h, cells/supernatant was collected for subsequent studies.
For transient transfection, HEK-293T and LO2 cells were seeded in a 6-well plate until 60–80% confluence. The volume/mass ratio of Exfect2000 Transfection Reagent (Vazyme) to DNA was 3:1. Transfection efficiency was evaluated using quantitative real-time polymerase chain reaction (qPCR) and Western blot. After transfection for 48 h, cells/supernatant was collected for subsequent studies.
9
Plasmid Transfection and Reporter Assay
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Expression vector pcDNA3.1(+) was purchased from Invitrogen (Carlsbad, CA, USA). A fast mutagenesis system kit was purchased from Transgen Biotech (Beijing, China). An Exfect 2000 transfection reagent was obtained from Vazyme Biotech (Nanjing, China). DMEM/F-12 1:1 medium was purchased from Thermo Fisher Scientific (Beverly, MA, USA). Reporter gene plasmids pGL4.29[luc2P/CRE/Hygro] and pGMLR-TK were purchased from Promega (Beijing, China). A dual-luciferase reporter gene assay kit was purchased from Beyotime Biotechnology (Shanghai, China). PMSF and protease inhibitors were purchased from Solarbio Life Science (Beijing, China). c-myc Rabbit mAb was obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG-HRP was purchased from Cell Signaling Technology (Boston, MA, USA).
10
Transfection of Mycoplasma-Contaminated MHCC97 Cells
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The American Type Culture Collection (ATCC) provided MHCC97 cells growing in RPMI-1640 medium with 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China). Mycoplasma contamination was found in these cell lines. The manufacturer's instructions were followed while transfecting cells using Exfect 2000 transfection reagent (Vazyme, NJ, China).
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