H9C2 cells were divided into four groups based on treatment: the Ad-GFP and Ad-UQCRC1 groups were infected with Ad-GFP or Ad-UQCRC1 as described above, whereas the Ad-UQCRC1+Zn2+ and Ad-UQCRC1+TPEN groups consisted of Ad-UQCRC1 cells treated with 1 μM ZnCl2 (Sigma-Aldrich) plus 4 μM pyrithione (Sigma-Aldrich) or 10 μM of the Zn2+-selective chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (Sigma-Aldrich), respectively, added 10 min before OGD/R injury.
Pyrithione
Manufactured by Merck Group
Sourced in Germany
Pyrithione is a chemical compound that functions as an antimicrobial agent. It is commonly used in various industrial and medical applications.
Automatically generated - may contain errors
Lab products found in correlation
Zncl2, by Merck Group (2 mentions)
Imiquimod, by Novus Biologicals (1 mentions)
Poly 1 c, by Novus Biologicals (1 mentions)
N n n n tetrakis 2 pyridylmethyl ethylenediamine tpen, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Zinc sulfate, by Merck Group (1 mentions)
Fluozin 3 am, by Thermo Fisher Scientific (1 mentions)
Zinc sulfate znso4, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
4 protocols using pyrithione
1
Zn2+ Modulation in OGD/R Injury
2
TLR Stimulation and Zinc Modulation
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All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. Medium composition for primary culture is described below. RAW 264.7 cells and HEK293T cells were grown in DMEM (Wako Chem., Osaka, Japan) supplemented with 10% FCS (Sigma-Aldrich, St Louis, MO), 100 U/ml penicillin, and 100 U/ml streptomycin (Wako Chem.). The TLR ligands poly I:C (Imgenex, San Diego, CA), LPS (Sigma-Aldrich), imiquimod (Imgenex), CpG-A (1585: Nihon Gene Research Laboratories, Sendai, Japan) and CpG-B (1668: Nihon Gene Research Laboratories) were used for TLR stimulation. N,N,N’,N’-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN; Sigma-Aldrich) was used for zinc chelation, and zinc loading of cells was performed by the addition of zinc sulfate (Sigma-Aldrich) and the ionophore pyrithione (Sigma-Aldrich). Doxycycline hydrochloride (Wako Chem.) was used at 1 μg/ml for the pTet-off system.
3
Measuring Intracellular Zinc in EL-4 Cells
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1 × 106 EL-4 T-cells were incubated with a final allicin concentration of 25 µM for 30 min with gentle shaking at 37 °C in the dark. Afterwards, cells were washed and loaded with 1 mL measurement buffer [32] (link) for 30 min, containing 1 µM FluoZin-3AM (Thermo Fisher, Germany) and again gently shaken at 37 °C in the dark. Control cells were not treated with allicin. Cells were washed, resuspended in measurement buffer and incubated at 37 °C for 10 min either left untreated or further supplemented with either N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 50 µM) to obtain minimal fluorescence or with a combination of zinc sulfate (ZnSO4) and pyrithione (100 µM/50 µM) (all Sigma-Aldrich, Germany) to obtain maximal fluorescence. Subsequent flow cytometry measurements were performed using FACSCalibur (BD, Germany). Calculation of intracellular labile zinc was performed as described before [33] (link) using the dissociation constant KD = 8.9 nM for the FluoZin-3/Zn2+ complex [34] (link).
4
High-Throughput Screening for Gαo Modulators
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HTS for mutant Gαo modulators was performed using the Gαo[E246K] protein and FDA Approved & Pharmacopeial Drug Library (HY-L066, MedChemExpress). Dimethyl sulfoxide (DMSO) or compounds in DMSO (12.5 μM) were mixed with Gαo[E246K] at 1 μM in a reaction buffer and BODIPY-GTP at 1 μM as described in the “GTP-binding and hydrolysis assay” section above. Reaction was carried out for 10 min.
To analyze the data generated by the HTS, two parameters were calculated: (i) binding constant (kbind) and (ii) maximal GTP uptake. For candidates affecting the kbind, the hits were picked if the compound modulated kbind by ≥2 SD of DMSO-treated wells. For candidates affecting the maximal BODIPY-GTP uptake, the hits were picked if the compound modulated the maximal GTP uptake by ≥3 SD of DMSO-treated wells. The hits were subsequently validated by performing the GTP-binding assay at 50 μM of compounds using both Gαo wild type and Gαo[E246K]. Validations were performed using commercially available sennosides (USP), ZPT (Sigma-Aldrich), ZnCl2 (Sigma-Aldrich), and pyrithione (Sigma-Aldrich).
To analyze the data generated by the HTS, two parameters were calculated: (i) binding constant (kbind) and (ii) maximal GTP uptake. For candidates affecting the kbind, the hits were picked if the compound modulated kbind by ≥2 SD of DMSO-treated wells. For candidates affecting the maximal BODIPY-GTP uptake, the hits were picked if the compound modulated the maximal GTP uptake by ≥3 SD of DMSO-treated wells. The hits were subsequently validated by performing the GTP-binding assay at 50 μM of compounds using both Gαo wild type and Gαo[E246K]. Validations were performed using commercially available sennosides (USP), ZPT (Sigma-Aldrich), ZnCl2 (Sigma-Aldrich), and pyrithione (Sigma-Aldrich).
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