One day after plating, DCDML cultures were transfected in M199 medium without BOTS or antibiotics using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). following the manufacturer's suggested protocol. Control experiments confirmed that the efficiency of transient transfection of DCDMLs is consistently ∼70%.43 (link) The SBE4-Luc reporter construct was provided by Bert Vogelstein (Johns Hopkins University; Addgene plasmid #16495). pcDNA3 plasmids encoding the following ErbB species were obtained from Addgene (Watertown, MA, USA): ErbB2-EGFP (#39321), originally from Martin Offterdinger; EGFR-GFP (#32751) from Alexander Sorkin; and ErbB4 (#29527) from Yardena Samuels.
Pcdna3 plasmid
Manufactured by Addgene
Sourced in United States
The pcDNA3 plasmid is a commonly used vector for gene expression. It contains a human cytomegalovirus (CMV) immediate-early promoter for high-level expression of the gene of interest in mammalian cells. The plasmid also includes a neomycin resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Egfr gfp, by Addgene (1 mentions)
Dharmafect, by Horizon Discovery (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Pcmv3 plasmids, by Sino Biological (1 mentions)
Keap1, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
7 protocols using pcdna3 plasmid
1
Transient Transfection of DCDMLs
2
Overexpression and Silencing of p16INK4a in Islet Cells
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The PCR product of the p16INK4a gene was cloned into pcDNA3 plasmids (Addgene, Cambridge, MA, USA) under the control of a cytomegalovirus promoter, and the plasmids were transfected into the primary cultured islet cells in the presence of Lipofectamine LTX (Thermo Fisher Scientific, Inc.). Small interfering RNA (siRNA) specific for p16INK4a pool (cat. no. 12578) and non-targeting siRNA controls were obtained from GE Dharmacon (Lafayette, CO, USA). Transfection with the siRNAs was performed using Dharmafect (GE Dharmacon).
3
Plasmid Constructs for BMP Signaling
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The pcDNA3.1 plasmids harboring the long form BMPR-2 and its kinase dead mutant BMPR-2K230R were kindly provided by Dr. Petra Knaus at Freie University Berlin (Berlin, Germany). The following plasmids were used in the current study: pCDNA3 plasmids harboring ALK2WT, ALK3WT and ALK6WT were purchased from Addgene (#80870, #80873 and #80882). pCMV3 plasmids harboring activin A receptor type 2A (ActR2A) and human activin A receptor type 2B (ActR2B) were obtained from Sino Biological Inc. The point mutations in human ALK2 (ALK2R206H), human ActR2A (ActR2AK219R) and ActR2B (ActR2BK217R) were made in-house using the QuikChange II site directed mutagenesis method. The sequences for mutagenic oligonucleotides are shown in Supplementary Table S1 . The mutations were confirmed by DNA sequencing.
4
Modulating Nrf2, KEAP1, and VHL Signaling
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Nrf2, KEAP1 and VHL siRNAs were purchased from Santa Cruz Biotechnology. To knock down genes, cell at 60% confluency were transfected with siRNA with Lipofectamine RNAiMAX Reagent (ThermoFisher), according to manufacturer’s protocol. Cisplatin was dissolved in water in a stock solution of 1mg/ml and add to cell culture to the final concentration of 10ug/ml. Dimethyl-bisphenol A (BPA) was dissolved in DMSO and added to the cell culture to the final concentration of 100uM. pcDNA3 plasmid expressing nondegradable HIF-1α(P402A/P564A) [43 (link)] was from Addgene (Constructs #18955). Plasmid were purified from bacteria and transfected into HepG2 cells with Lipofectamine 3000. An empty pcDNA3 plasmid was used as a control.
5
Wound Healing Assay with HIF1A Overexpression
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Cells were seeded onto 6-well plates in equal number and allowed to rest until confluent. A wound of equal width was made with a sterile 10 μL pipette tip along the middle of the well. Media was aspirated and replaced with media containing 75 uM GSKLSD1 or DMSO. Time points were taken every 6 h until the wound closed. Cells were transfected with a pcDNA3 plasmid expressing a constitutively active mutant HIF1A with P402A and P564A substitutions (Addgene) [31 (link)].
6
Recombinant Expression of Mutant HIF2A
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A pcDNA3 plasmid (Addgene, Cambridge, MA; plasmid 18950 [7 (link)]) containing the human HIF2A coding sequence was used for wild-type HIF2A expression analysis. E549K-mutant HIF2A was cloned and inserted into an expression plasmid using a Quikchange Lightning Site-directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Sanger sequencing was used to confirm successful insertion.
7
ISL1 Overexpression in Breast Cancer Cells
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The full-length sequence of ISL1 was amplified from cDNA of 293 cells and cloned into the pcDNA3 plasmid (Addgene, Inc., Cambridge, MA, USA). For ISL1 overexpression, 1×106 MDA-MB-231 and MDA-MB-468 cells were transfected with 2 μg pcDNA3-ISL1 using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.), while cells transfected with empty vector served as controls. After 24 h, the cells were subjected to following experiments.
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