After the pre-experiment, Kasumi-1 cells were dispensed at 200 mL on 96-well plates at a density of 10 x 10 4 cells/mL at 37°C in a 5% CO 2 humidified atmosphere. Next, 10 mg/ mL DNR (Sigma Co., St. Louis, MO, USA), 10 mM ZnPPIX (Sigma), and 10 mM ZnPPIX + 10 mg/mL DNR were added to the cells for 24, 48, and 72 h. A blank control group was established, and the experiment for each group was performed in triplicate. Next, 20 mL 5 mg/ mL MTT (Solarbio Science & Technology Co., Ltd., Beijing, China) was added to each well, followed by incubation for 4 h at 37°C. Finally, 150 mL dimethyl sulfoxide was added to each well, after which absorbance was read at 570 nm on a microplate reader. The survival rate was calculated according to the equation: survival rate (%) = A drug group /A control group x 100%.
Znppix
Manufactured by Merck Group
Sourced in United States
ZnPPIX is a zinc-containing compound that can be used as a laboratory reagent. It has potential applications in various biochemical and biophysical research areas.
Automatically generated - may contain errors
Lab products found in correlation
Hemin, by Merck Group (3 mentions)
P0674, by Merck Group (1 mentions)
Primary antibodies for western blot analysis, by Cell Signaling Technology (1 mentions)
Sh 4 54, by Selleck Chemicals (1 mentions)
Imatinib, by Novartis (1 mentions)
Nilotinib, by Novartis (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
H9c2 cardiomyoblast, by American Type Culture Collection (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
β actin rabbit antibody, by Cell Signaling Technology (1 mentions)
Sybr green pcr master mix, by Promega (1 mentions)
Rat igg2a isotype control pe, by Thermo Fisher Scientific (1 mentions)
Rabbit ho 1 antibody, by Abcam (1 mentions)
Tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Amv transcriptase, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
18 protocols using znppix
1
MTT Assay for Cytotoxicity Evaluation
2
Biochemical Assay for Inflammatory Markers
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ZnPPIX was bought from Sigma. TNF-α and HMGB1 concentrations in the serum or culture media were determined by ELISA using commercial ELISA kits obtained from Bio-Legend (San Diego, CA, USA). Data were analyzed by Student’s t-test. A p-value <0.05 was considered statistically significant.
3
Carbon Monoxide and Zinc Protoporphyrin IX Protocol
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Stock solution (at 10 mM) of COMR-2 and ZnPPIX (Sigma) were dissolved in DMSO (dimethylsulphoxide). Final use concentration solutions were made by diluting stocks with sterilized medium. In this study, 10, 20, and 40 μM were used as final concentrations for CORM-2, and 10 μM for ZnPPIX. An equivalent concentration of DMSO was added in the plates as control. Seeds were sterilized and germinated on half-MS medium supplemented with or without chemicals. All the treatments have at least three replicates.
4
Hyperoxia-Induced Lung Injury Protocol
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Figure 1A shows the structure of ZJ01, which was synthesized as previously reported [12 (link)]. ZJ01 (10mg/kg) or vehicle was administrated intraperitoneally at 1 h after the hyperoxia challenge, and an additional dose of ZJ01 or vehicle was administrated every 24 h. On review of the protocols from a previous study [12 (link)], a preliminary study was performed to determine the dose of ZJ01 used in this study (data not shown).
ZJ01 was freshly dissolved in the vehicle (DMSO: normal saline (v/v)=5: 95). Thirty minutes before ZJ01 treatment, the HO-1 inhibitor, ZnPP IX (50 mg/kg−1), was given by intraperitoneal injection (Sigma Chemical Co. St. Louis, MO, USA.) [13 (link)]. An additional dose of ZnPP IX (50 mg/kg) was administrated every 24 h. The animals were then euthanized under anesthesia by 50 mg/kg−1 of pentobarbitone by intraperitoneal injection at 72 h after room air or hyperoxia stimulation. The left lung was removed for the measurement of lung edema. The right lung was harvested and immediately frozen in liquid nitrogen for further study.
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ZJ01 was freshly dissolved in the vehicle (DMSO: normal saline (v/v)=5: 95). Thirty minutes before ZJ01 treatment, the HO-1 inhibitor, ZnPP IX (50 mg/kg−1), was given by intraperitoneal injection (Sigma Chemical Co. St. Louis, MO, USA.) [13 (link)]. An additional dose of ZnPP IX (50 mg/kg) was administrated every 24 h. The animals were then euthanized under anesthesia by 50 mg/kg−1 of pentobarbitone by intraperitoneal injection at 72 h after room air or hyperoxia stimulation. The left lung was removed for the measurement of lung edema. The right lung was harvested and immediately frozen in liquid nitrogen for further study.
5
Reagents for Heme-Signaling Pathway
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Hemin and ZnPPIX were purchased from Sigma (Saint Quentin Fallavier, France) and Frontier Scientific (Distributor: Inochem, Ltd, Carnforth, Lancashire, United Kingdom), respectively. QC‐15 was a kind gift from Prof Kanji Nakatsu from Queen’s University, Kingston, Ontario, Canada. All other reagents were purchased from Sigma unless otherwise stated.
6
HKs Oxidative Stress Response
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HKs were acquired from ScienCell Research Laboratories (catalog #6520) and cultured in fibroblast medium. All cells on the hydrogel substrate and on common culture dishes were cultured for 24 h before detection or drug treatment. HKs were treated with SF (50 μM, 4 h) (Sigma) in the presence or absence of 10 μM Znpp IX (a specific inhibitor of HO-1, 1 h) (Sigma). For oxidative stress analysis, HKs were treated with 200 μM H2O2 for 1 h with or without 50 μM SF for 4 h and 10 μM Znpp IX for 1 h. The treatment concentrations used in our study were evaluated and optimized in our primary experiments. Cells from the third to fifth passages were utilized in this study.
7
Cecal Ligation and Puncture Sepsis Model
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Mice were randomly divided into four groups: Sham group, CLP group, Dex group (CLP + Dex) and Dex + zinc protoporphyrin (CLP + Dex + ZnPP). Following CLP or sham surgery, intraperitoneal injections of 40 µg/kg Dex or saline were immediately administered once. Znpp IX (40 mg/kg) was injected via intraperitoneal administered 1 h before the CLP operation (20 (link)). Znpp IX (Sigma-Aldrich; Merck KGaA) was dissolved in 0.2 M sodium hydroxide and adjusted to a pH of 7.4 (21 (link)).
8
Visualizing Heme Pathways with ZnPPIX
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Uptake of a fluorescent metalloporphyrin, zinc mesoporphyrin, as a fluorescent haem analogue, was used to characterize haem intracellular pathways similarly to a previously published protocol [39 (link)]. A 20 mM stock solution of ZnPPIX (SigmaAldrich 691550-M) was dissolved in 100 mM NaOH and further diluted in sterile PBS before serving to ticks. Five microlitres of 100 µM ZnPPIX were introduced (2 h at 37°C) through 10 µl microcapillaries (Sigma-Aldrich P0674) into I. ricinus ticks, pre-fed 5 days on a guinea pig. Ticks were then rested for 24 h at 21°C. ZnPPIX was visualized in the dissected midguts of partially engorged ticks subjected as described below to RNA interference. The fluorescent images were obtained using a fluorescence microscope Olympus BX63 (filter U-FGWA, emission 575–625 nm) and DP74 digital camera.
9
Modulation of Inflammation by Heme and Zinc
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Animals were divided into 10 groups (n = 10 each) as follows: Control (C), LPS (L), LPS + Hemin (LH), LPS + Znpp (LZ), LPS + Hemin + Znpp (LHZ), Hemin (H), Znpp (Z), Hemin + Znpp (HZ), LPS + NaHCO3 (LNaHCO3) and LPS + NaOH (LNaOH). Drugs were injected via the tail vein: C received 1 ml of 0.9% saline vehicle; LPS (LPS, Sigma, USA) was injected at a dose of 5 mg/kg dissolved in 1 ml of 0.9% saline vehicle; Hemin (Hemin, Sigma, USA) was injected at the dose of 100 mg/kg dissolved in 1 ml of NaOH; and zinc protoporphyrin-IX was injected at a dose of 10 μmol/kg dissolved in 1 ml of NaHCO3 (ZnPP-IX, Sigma, USA).
10
Evaluation of Anti-Leukemia Compounds
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Imatinib (STI571) and nilotinib (AMN107) were kindly provided by Novartis Pharma AG (Basel, Switzerland). HO-1 inhibitor ZnPPIX were purchased from Sigma (St. Louis, MO, USA). AZ960 was kindly provided by AstraZeneca R&D (Waltham, MA). SH-4-54 was from Selleck Chemicals (Houston, TX). Primary antibodies for western blot analysis were obtained from Cell Signaling Technology (Beverly, MA), and secondary antibodies were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Primary antibodies used in this study including anti-HO-1, anti- Cleaved Caspase-3, anti-Ikaros, anti-STAT5, anti-phosphorylated-STAT5 (pSTAT5), anti- Actin and anti-LaminB.
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