We designed a wild-type JUP CDS plasmid with HIS-tag in a pcDNA3.1+ vector. The R577C-JUP missense mutation was engineered into the vector using the Takara MutanBEST Kit (Takara Bio, Otsu, Shiga, Japan). AC16 cells were transfected with HIS-JUP-pcDNA3.1+ (WT and p.R577C) by using Lipofectamine™ 2000 CD Transfection Reagent (Thermo Fisher Scientific), following the manufacturer's instructions.
Lipofectamine 2000 cd transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine™ 2000 CD Transfection Reagent is a cationic lipid-based transfection reagent designed for efficient delivery of nucleic acids into a variety of cell types, including hard-to-transfect cell lines. The reagent forms complexes with DNA, RNA, or other nucleic acids, which are then taken up by the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Takara mutanbest kit, by Takara Bio (1 mentions)
Mir nc mimic, by GenePharma (1 mentions)
Mir nc inhibitor, by GenePharma (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Nebuilder hifi dna assembly, by New England Biolabs (1 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
8 protocols using lipofectamine 2000 cd transfection reagent
1
Engineering JUP Missense Mutation
2
Transfection of miRNA mimics and inhibitors
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MiR-205-5p mimic and its scrambled control (miR-NC mimic), miR-205-5p inhibitor and inhibitor control (miR-NC inhibitor) and were purchased from GenePharma Co. Ltd. (Shanghai, China). The sequences of the oligonucleotides used were shown in Table 2 . FLSs (4×105 cells per well) were seeded into 6-well plates and cultured as described above. Cells were transfected by Lipofectamine™ 2000 CD Transfection Reagent (12566014, Thermo Scientific, MA, USA) according to the instructions. The final concentrations of miRNAs were 50 nM, and were transfected into cells using 0.25 % lipofectamine 2000 for 48 h at 37°C.
3
Validation of miR-205-5p Targeting MDM2
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FLSs (4×105 cells per well) were seeded into 6-well plates and cultured as described above. The bioinformatics analysis softwares, such as miRwalk, targetscan, miRDB, miRTarbase were used to predict the genes (such as MDM2) targeting with miR-205-5p. The putative binding sites of miR-205-5p on the MDM2 3’-UTR were predicted using the TargetScan 7.0 (http://www.targetscan.org/vert_70/) online tool. The MDM2 3’-UTR sequences were introduced into the luciferase reporter vector (pGL3-Basic) to construct wild-type (WT) luciferase reporter plasmids (MDM2 WT), and were mutated to construct mutant (MUT) luciferase reporter plasmids (MDM2 MUT). FLSs were co-transfected with 0.5 µg/µl luciferase reporter plasmids, 50 nM miR-489 mimics or inhibitors, and their negative control using Lipofectamine™ 2000 CD Transfection Reagent (12566014, Thermo Scientific, MA, USA). After 48 h, cells were collected and measured using a luciferase reporter assay system (Promega, WI, USA). The dual-luciferase activity of the target gene was normalized to Renilla luciferase activity. The reported data represent the average of three independent transfection experiments performed in triplicate.
4
Generation and Validation of RCas9 Cell Lines
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The RCas9 vector was purchased from Addgene (LentiRCas9-CUG, #104183) and sgCUG sgRNA sequence was replaced by TERRA antisense or control sequences using NEBuilder HiFi DNA assembly (NEB, cat#E2621L). U2OS cells were transfected with RCas9 plasmids using Lipofectamine™ 2000 CD Transfection Reagent (Thermo Fisher Scientific, cat#12566014) and selected by 1 µg/ml puromycin for one week. Survival cell colonies were picked and maintained in 0.5 µg/ml puromycin DMEM medium. Positive clones were further confirmed by PCR. Cells were lysed in the lysis buffer (2X Roche PCR buffer, 0.45% NP 40, Tween 20) at 55 °C for 2 h and inactivated at 95 °C for 15 min. Cell lysates were subjected to PCR. Primers for RCas9 (forward primer (5’-GTT TAA GAG CTA TGC TGG AAA C-3’), reverse primer (5’-CCT AGC TAG CGA ATT CGC GC-3’)) was used to detect 200 bp sequences located downstream of sgRNA. Plasmids and cell lines generated in this study are available from the corresponding author upon request.
5
Transfection of PDA Cells with UTX and GATA siRNAs
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The plasmid pCMV-HA-UTX was generated by Addgene. Small interfering RNAs (siRNAs) synthesized by Invitrogen were as follows: UTX siRNA (#1: 5′-gcaaauguuccaguguauagguuua-3'; stealth#2: 5′-ucaguuagcuuugguugacuguaau-3′) and GATA SiRNA (#1:5′-guggacucuacaugaaacutt-3’; #2, 5′-gcucugguaauagcaauaatt-3′). Negative control siRNA (Invitrogen) and control pCMV vectors were used. Plasmids and siRNAs were transfected into PDA cells using Lipofectamine 2000 CD transfection reagent (Invitrogen). For transient transfection, cells were transfected with plasmids or siRNA at different doses as indicated for 48 h before functional assays.25 (link),42 (link),43 (link)
6
Engineered SFTPA2 Missense Mutation
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The wild‐type SFTPA2 CDS (NM_001098668) with a C‐terminal HIS‐tag in the pEnter was designed by us. The p.N207Y‐SFTPA2 missense mutation was constructed into the above vector using the QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies). Sanger sequencing was applied to check the constructs. A549 cells were transiently transfected with 2 μg SFTPA2‐HIS‐pEnter plasmids (WT and/or mutation) using Lipofectamine™ 2000 CD Transfection Reagent (Invitrogen™), according to the manufacturer's instructions and cultured for 72 hr.
7
Plasmid Transfection and siRNA Knockdown in PDAC Cells
8
Triad1 and MDM2 Overexpression and Knockdown in Astrocytes
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Prior to transfection, plasmids overexpressing Triad1 and MDM2, their negative control (NC), shRNAs targeting Triad1 (sh-Triad1), PTN (sh-PTN) and MDM2 (sh-MDM2), and NC of shRNA (sh-NC) were synthesized in RIBOBIO. For transfection, astrocytes were cultured in a 6-well plate until 90% confluence was reached. Then, the aforementioned plasmids and shRNAs were separately transfected into the cells with the help of Lipofectamine 2000 CD transfection reagent (12566014, Invitrogen). After 48 h of transfection, the cells were collected for later use.
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