HH-N25 was synthesized through an established protocol in our lab,39 (link) while paclitaxel was procured from Selleckchem (Houston, TX, USA). TOPI and TOPII, supercoiled pRYG DNA, supercoiled pHOT1 DNA, camptothecin, and etoposide were purchased from TopoGEN. All other reagents and chemicals including DMEM (Gibco-Invitrogen, Grand Island, NY, USA), Matrigel (BD Biosciences, USA), Cremophor EL (Sigma, St. Louis, MO, USA), FBS (Hyclone, Logan, UT, USA), dimethylacetamide (DMA; Sigma), estradiol cyclopentyl propionate (Estol-depot injection, Astar, Taipei City, Taiwan), and phosphoric acid (Wako, Japan) were procured from reputable manufacturers.
Supercoiled phot1 dna
Manufactured by TopoGEN
Supercoiled pHOT1 DNA is a plasmid DNA product suitable for various molecular biology applications. It is a circular, double-stranded DNA molecule that is maintained in a supercoiled state, which is a compact and stable DNA conformation.
Automatically generated - may contain errors
Lab products found in correlation
Phosphoric acid, by Fujifilm (1 mentions)
Camptothecin, by TopoGEN (1 mentions)
Etoposide, by TopoGEN (1 mentions)
Paclitaxel, by Selleck Chemicals (1 mentions)
Dimethylacetamide dma, by Merck Group (1 mentions)
Cremophor el, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Loading stop buffer, by TopoGEN (1 mentions)
Mercuric chloride, by Merck Group (1 mentions)
Ammonium sulfamate, by Merck Group (1 mentions)
Sulfanilamide, by Merck Group (1 mentions)
Sds kcl precipitation assay kits, by TopoGEN (1 mentions)
Propylamine propylamine nonoate ppno, by Cayman Chemical (1 mentions)
Sodium nitrite, by Merck Group (1 mentions)
Pifithrin α, by Merck Group (1 mentions)
Nitric oxide assay kit, by Thermo Fisher Scientific (1 mentions)
Antibody to human topo 1, by TopoGEN (1 mentions)
Human topo 1, by TopoGEN (1 mentions)
N 1 naphthyl ethylenediamine dihydrochloride, by Merck Group (1 mentions)
3 protocols using supercoiled phot1 dna
1
Synthesis and Procurement of Anticancer Agents
2
Topoisomerase I Activity Assay
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Top1 activity (TopoGEN Inc.) was measured by relaxation of supercoiled pHOT-1 DNA, following the manufacturer’s protocol. Briefly, 1 μl of test solution, 2 μl of 10× Top1 “reaction” buffer (TopoGEN Inc.), and 125 ng of supercoiled pHOT-1 DNA (TopoGEN Inc.) were combined in molecular biology–grade dH2O at a final assay volume of 20 μl and incubated at 37°C for 30 min. After incubation, 4 μl of 5× loading stop buffer (TopoGEN Inc.) was added and mixed by vortex. The reaction products were separated on a 1% TBE/agarose gel (no EtBr) and stained with EtBr, and the bands were imaged. Enzyme activity was quantified by measuring the intensity of the relaxed pHOT-1 bands by densitometry using ImageJ (fig. S13).
3
Nitric Oxide Regulation of Topoisomerase I
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Camptothecin was a gift of the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program of NCI, NIH. Human topo I, antibody to Human topo I, Supercoiled pHOT1 DNA, and SDS/KCl precipitation assay kits were obtained from Topogen (Port Orange, FL). The nitric oxide donor, propylamine propylamine nonoate (PPNO), was obtained from Cayman Chemicals (Ann Arbor, MI). A stock solution of PPNO was prepared in 0.2 N NaOH and was stored at -80°C. Pifithrin-α, cysteine, sodium nitrite, ammonium sulfamate, mercuric chloride, sulfanilamide and N-1-naphthylethylenediamine dihydrochloride were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Primary antibodies for the analysis of bcl2, Bax, p53, and p21 were from Santa Cruz Biochemicals (Dallas, TX). A nitric oxide assay kit was purchased from Thermo Scientific (Waltham, MA). Anti-s-nitroso-cys (SNO-Cys) was purchased from Alpha Diagnostic International, San Antonio, TX, USA)
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