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Tmpyp4

Manufactured by Merck Group
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Sourced in Germany, United States

TMPyP4 is a chemical compound that functions as a photosensitizer. It is used as a research tool in various scientific applications.

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Lab products found in correlation

Braco 19, by Merck Group (6 mentions) Sypro orange, by Thermo Fisher Scientific (4 mentions) Pyridostatin, by Merck Group (3 mentions) Tmpyp2, by Frontier Scientific (2 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions) Congo red, by Merck Group (2 mentions) Phendc3, by Merck Group (2 mentions) Oligonucleotide, by Integrated DNA Technologies (2 mentions) Purexpress in vitro protein synthesis kit, by New England Biolabs (1 mentions) Spark microplate reader, by Tecan (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Conventional petri dishes, by BD (1 mentions) Dmem glutamax, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Euroclone (1 mentions) Rna oligonucleotide, by Merck Group (1 mentions) K2hpo4, by Merck Group (1 mentions) Dna oligonucleotides, by Merck Group (1 mentions) Kh2po4, by Merck Group (1 mentions)

22 protocols using tmpyp4

1

Murine Myoblast C2C12 Differentiation

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Murine myoblast C2C12 according to [29 (link)] were cultured on conventional Petri dishes (BD Falcon) at 37°C and 5% CO2 in DMEM GlutaMAX (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (FBS, EuroClone), penicillin (100 IU/mL, Gibco), and streptomycin (100 mg/mL, Gibco). All the experiments were conducted for 10 days starting from a cellular confluence of 50%, to obtain a high degree of differentiation, and cells were divided into two experimental groups: the control group cultured in growth medium and the treated group cultured in growth medium with the addition of TMPyP4 (Sigma-Aldrich) to a final concentration of 12 μM. Medium was changed every day as well as fresh addition of TMPyP4.
Testa S., D'Addabbo P., Fornetti E., Belli R., Fuoco C., Bernardini S., Cannata S., Frezza D, & Gargioli C. (2018). Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction. Oxidative Medicine and Cellular Longevity, 2018, 7640272.
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2

Synthesis and Expression of Chimeric Proteins

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BG4,31 (link) conjugated with FluoProbes647H (Cy5-BG4), was obtained from Absolute Antibody (product number Ab00174-1.1). TMPyP4 was obtained from Sigma-Aldrich (catalog number 613560). N-terminal glutathione S-transferase (GST) tagged human nucleolin IGF2, CNBP, and helicase plasmids were synthesized by GenScript. Purified, recombinant bovine DHX3641 (link) was provided as a gracious gift by the Ferré-D’Amaré Lab (National Institutes of Health, Bethesda). The sequences of all proteins used are listed in Table S1. All chimeric proteins were expressed via in vitro translation (IVT) reactions using the PURExpress In Vitro Protein Synthesis Kit (NEB) as described previously.23 (link) For all IVT reactions, 288 ng of plasmid was added to 80 μL of a IVT mixture, and reactions were carried out at 37 °C for 2 h. Expression of all protein constructs was confirmed via Western blot (Figure S2).
Ray S., Tillo D., Boer R.E., Assad N., Barshai M., Wu G., Orenstein Y., Yang D., Schneekloth JS J.r, & Vinson C. (2020). Custom DNA Microarrays Reveal Diverse Binding Preferences of Proteins and Small Molecules to Thousands of G-Quadruplexes. ACS chemical biology, 15(4), 925-935.
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3

G-Quadruplex Stabilization and Fluorescence Detection

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BRACO-19, PDS and TMPyP4 powders were purchased from Sigma, MedChemExpress and TCI, respectively. The compounds were diluted in 10 mM Tris HCl (pH = 7.4) and 100 mM KCl buffer. The sequences of G-quadruplex and G-mut were listed in Table 2. Synthetic RNA oligonucleotides in powder were diluted to 2 μM final concentration in 10 mM Tris HCl (pH = 7.4) and 100 mM KCl buffer, heated at 95°C for 5 min and slowly cooling down to room temperature. The RNA solution was incubated at 4°C overnight in the absence or presence of compounds. ThT powder was bought from Aladdin Industrial Corporation. Oligonucleotides and ThT were mixed at 1:0.5 M ratio to a final concentration of 1 and 0.5 μM, respectively. Fluorescence emission was recorded at 495 nm after excitation at 425 nm using a TECAN SPARK microplate reader (30 (link)).
Lv L, & Zhang L. (2022). Characterization of G-Quadruplexes in Enterovirus A71 Genome and Their Interaction with G-Quadruplex Ligands. Microbiology Spectrum, 10(3), e00460-22.
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4

Binding Assay for G-quadruplex Ligands

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Small-molecule ligands, namely Braco-19 and TMPyP4, were purchased from Sigma Aldrich Chemicals, dissolved in water or DMSO, respectively, for preparation of stock solutions, and stored at 4°C until further use. Other reagents and chemicals including KCl, KH2PO4, K2HPO4, DMSO, and D2O were procured from Sigma Aldrich Chemicals or HiMedia Laboratories. The DNA oligonucleotides and the RNA oligonucleotides used in the studies were obtained from Sigma Aldrich Chemicals and IDT Technologies, respectively (Table S8).
Majee P., Pattnaik A., Sahoo B.R., Shankar U., Pattnaik A.K., Kumar A, & Nayak D. (2021). Inhibition of Zika virus replication by G-quadruplex-binding ligands. Molecular Therapy. Nucleic Acids, 23, 691-701.
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5

Synthesis of IQ3A Derivatives

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7-carboxylate indolo[3,2-b]quinoline tri-alkylamine derivatives 1a-d and 2a-d (IQ3A) were synthesized as described in S1 Text. 5-fluorouracil (5-FU) and TMPyP4 were purchased from Sigma-Aldrich, St Louis, MO, USA.
Brito H., Martins A.C., Lavrado J., Mendes E., Francisco A.P., Santos S.A., Ohnmacht S.A., Kim N.S., Rodrigues C.M., Moreira R., Neidle S., Borralho P.M, & Paulo A. (2015). Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives. PLoS ONE, 10(5), e0126891.
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6

MTT Assay for Cell Proliferation

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Cell proliferation assay was performed in 96 well plate by incubating HEK293T cells (seeding density = 1 × 104 cells/well) in the presence of multiple doses of TMPyP4 (Sigma) or Pyridostatin (Sigma). After 24 h, cells were exposed to MTT (3-(4,5-Dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) reagent for 1 h. The medium was replaced with 100 μl dimethylsulfoxide (DMSO) and optical density measured at 570 nm (Additional file 1, Figure S1).
Kumar S., Choudhary D., Patra A., Bhavesh N.S, & Vivekanandan P. (2020). Analysis of G-quadruplexes upstream of herpesvirus miRNAs: evidence of G-quadruplex mediated regulation of KSHV miR-K12–1-9,11 cluster and HCMV miR-US33. BMC Molecular and Cell Biology, 21, 67.
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7

Cell Culture and Treatment Protocol

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U2OS and HEK 293T cells purchased from the American Type Culture Collection (ATCC; Manassas, USA) were grown in DMEM (Corning, New York, USA). HeLa cells purchased from ATCC were grown in 1640 medium (Corning). All media were supplemented with 10% fetal bovine serum (Lonsera, Canelones, Uruguay) and 1% penicillin-streptomycin (HyClone, Logan, USA, and cells were cultured at 37°C with 5% CO
2. Cells were treated with 2 mM hydroxyurea (HU; Sigma-Aldrich, Darmstadt, Germany), 10 μM pyridostatin (PDS; Sigma-Aldrich), or 50 μM TMPyP4 (Sigma-Aldrich) for 24 h.
Li T., Tang L., Kou H, & Wang F. (2022). PRIMPOL competes with RAD51 to resolve G-quadruplex-induced replication stress via its interaction with RPA: PRIMPOL resolves G4-induced replication stress. Acta Biochimica et Biophysica Sinica, 55(3), 498-507.
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8

Cytotoxicity Evaluation of TMPyP4 and Cisplatin

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TMPyP4 (Item Number: 323497) and LPS (Item Number: L6386) were purchased from Sigma‐Aldrich with purity ≥97%. Cisplatin was from Selleck (Item number: S1166). TNF‐α was from Peprotech (Item number: 315‐01A). TMPyP4 was dissolved in water, and Cisplatin was dissolved in dimethyl sulfoxide (DMSO) for storage and further diluted to final concentrations.
Chen J., Jin X., Mei Y., Shen Z., Zhu J., Shi H., Wang M., Zheng X, & Liang G. (2021). The different biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of osteosarcoma are attributed to G‐quadruplex. Cell Proliferation, 54(9), e13101.
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9

SYBR Green PCR Assay with GC-R1 and GC-R2

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All PCR reactions were performed on a 7500 Fast Real-time PCR System (Applied Biosystems) using SYBR Green PCR master mix (Applied Biosystems) with 0, 5, 10 or 25nM of either TMPyP4 (Sigma-Aldrich) or TMPyP2 (Frontier Scientific). The data shown are the mean of three independent trials with error bars representing the standard error of the mean. Both wild-type and mutant oligonucleotide template strands were the same as those used in the hnRNP K pull-down experiments (see above).
Primer annealing to the GC-R1 wild-type and mutant forward strand: 5′-AGTCAGTCCGTCAAAG-3′
Primer annealing to the GC-R1 wild-type and mutant reverse strand: 5′-AGTCAGTCGCAGGCGT-3′
Primer annealing to the GC-R2 wild-type and mutant forward strand: 5′-AGTCAGTCGTGCCTGA-3′
Primer annealing to the GC-R2 wild-type and mutant reverse strand: 5′-AGTCAGTCTCTCGTCG-3′
Banerjee K., Wang M., Cai E., Fujiwara N., Baker H, & Cave J.W. (2014). Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure. Nature communications, 5, 5769.
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10

Oligonucleotide Characterization and Fluorescent Ligand Binding

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All oligonucleotides (Table S1) were purchased from Invitrogen (Beijing, China), purified by PAGE. The stock solutions of the oligonucleotides were prepared by dissolving oligonucleotides directly into 20 mM Tris-HCl buffer (pH 7.0) and annealed in a thermocycler (first heating at 90 °C for 2 min, then cooled down to room temperature slowly). The ligands PDS (NO. SML0678, Sigma), TmPyP4 (NO. 613560, Sigma), San (NO. IS0040, Solarbio) and RHPS4 (NO.B6186, APExBIO, USA) were used as received without further purification. Cell nucleus staining dyes propidium iodide (PI) and SYTO®59 were all obtained from Thermo Fisher Scientific Company. All other ordinary solvents and chemical regents stock solution of IMT (10 mM) was prepared in methanol. Ultrapure water, prepared by Milli-Q Gradient ultrapure water system (Millipore), was used in all experiments.
Zhang S., Sun H., Yang D., Liu Y., Zhang X., Chen H., Li Q., Guan A, & Tang Y. (2019). Evaluation of the selectivity of G-quadruplex ligands in living cells with a small molecule fluorescent probe. Analytica Chimica Acta: X, 2, 100017.
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