Hoechst 33258, and calcium ionophore III were obtained from Sigma (St Louis, MO, USA). Acridine orange (AO)/ethidium bromide (EB) double stain assay kit, L-glutathione (Reduced) and tunicamycin were purchased from Solarbio Science & Technology Co. (Beijing, China). Protein A/G-agarose was from Biogot Technology Co. (Nanjing, Jiangsu, China). Lipofectamine RNAiMAX and Lipofectamine 2000 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Pronase was from Roche Diagnostics GmbH (Mannheim, Germany) and recombinant histone H3 was purchased from Active Motif (CA, USA). LysoTracker, MitoSox, MitoTracker Green, and MitoTracker Red were purchased from Thermo Fisher Scientific (Waltham, Ma, USA). ER-Red and DIOC6 (3) were acquired from Nanjing KeyGen Biotech Co. (Nanjing, Jiangsu, China). Fluo 3-AM special packaging was purchased from Dojindo Laboratorie (Kumamoto-ken, Kyushu, Japan). Thapsigargin was obtained from ACROS Organics (Belgium). LysoSensorTM Yellow/Blue DND-160 (PDMPO) was purchased from Yeasen (Shanghai, China). DO2, NaOD, and DCl were acquired from J&K Scientific (Beijing, China). R18 peptide was obtained from Enzo Life Sciences (New York, USA).
Tunicamycin
Manufactured by Solarbio
Sourced in China
Tunicamycin is a naturally occurring antibiotic that inhibits the first step in the biosynthesis of N-linked glycoproteins. It is commonly used in cell biology research to study the role of glycosylation in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
N acetyl l cysteine, by Beyotime (2 mentions)
L glutathione reduced, by Solarbio (1 mentions)
Thapsigargin, by Thermo Fisher Scientific (1 mentions)
R18 peptide, by Enzo Life Sciences (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Pronase, by Roche (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Paclitaxel, by Solarbio (1 mentions)
Paclitaxel, by Apexbio (1 mentions)
Tunicamycin, by Thermo Fisher Scientific (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Recombinant human il 1β, by Sangon (1 mentions)
Paclitaxel, by Thermo Fisher Scientific (1 mentions)
10 protocols using tunicamycin
1
Cellular Imaging: Protocols and Reagents
2
Jurkat-Tumor Cell Co-culture Assay
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Jurkat T cells were activated by Dynabeads Human T-Activator CD3/CD28(11161D, Gibco, USA), and were then co-cultured with 5-8F or CNE1 cells pretreated with SB431542 (20μM) (HY-431542, MCE, USA) or tunicamycin (15μg/ml) (T8480, Solarbio, China) for 48h at 3:1 (Jurkat: 5-8F/CNE1) ratio for 6h. The death rate of tumor cells was detected by CFSE/PI Double Stain Kit (BB4214, Bestbio, China). The medium supernatant of the co-culture system was collected for detecting the level of IL-2 by the ELISA kit (70-EK102, MultiSciences, China) according to manufacturer’s instructions.
3
Cell Viability Evaluation by CCK-8 Assay
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The CCK-8 assay (Dojindo Molecular Technologies, Inc.) was performed to test cell viability. Following treatment with 8 nM paclitaxel (APeXBIO Technology LLC), 20 ng/ml recombinant human IL-1β (Sangon Biotech Co., Ltd.), 0.1 µg/ml tunicamycin (Beijing Solarbio Science & Technology Co., Ltd.), paclitaxel + IL-1β, paclitaxel + tunicamycin and blank control, then 2x103 A549 cells were seeded into 96-well plates and incubated at 37˚C with 5% CO2 for 48 h. At 0, 12, 24 and 48 h, 10 µl CCK-8 reagent was added to each well and incubated with the cells for 2 h. Absorbance was measured at 450 nm by a microplate reader (Thermo Fisher Scientific, Inc.).
4
Apoptosis Induction in LUAD Cells
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Cells were treated with 10μg/mL of tunicamycin (#T8480, Solarbio, China) and then treated with 1.0 μM GSK2606414 (the PERK inhibitor, Selleck Chemicals, USA), 50 μM STF083010 (the IRE1 inhibitor, MedChemExpress, USA), 20 μM Z-VAD-FMK (apoptotic inhibitor, MedChemExpress, USA), or DMSO for 48 h. tunicamycin, a natural antibiotic, triggers cancer cell apoptosis 14 (link) and has been diffusely applied to the study of tumor cell apoptosis and programmed cell death. All cells were treated with Annexin V-APC and 7-AAD (MultiSciences, China) at room temperature for 30 mins. The percentage of apoptotic LUAD cells was detected by flow cytometry (Becton Dickinson, USA).
5
Investigating ER Stress Responses
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The antibodies used in our experiments are listed in Table S1 . Tunicamycin (TM, ER stress inducer) was purchased from Solarbio (Beijing, China) and stocked at concentration of 2 mm in dimethyl‐sulfoxide (DMSO). MG132 (proteasome inhibitor), and cycloheximide (CHX, protein synthesis inhibitor) was purchased from Calbiochem (Darmstadt, Germany) and stocked at concentration of 20 mm and 50 mg·mL−1 in DMSO, respectively. Radezolid (RRBP1 inhibitor) was purchased from APExBIO (Houston, TX, USA) and stocked at the concentration of 10 mm in DMSO. 0.5, 1, and 2 µm TM were used for 48 h or 2 µm TM was used for 24, 48, and 72 h to induce ER stress. Ten micromolar MG132 was used for 6 h prior to harvesting the cells, and 50 μg·mL−1 CHX was used for indicated time to detect protein degradation. Ten micromolar Radezolid was used to inhibit RRBP1 expression.
6
Modulating Oxidative and ER Stress in Hypoxic Hepatocytes
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The mouse hepatocyte cell line AML12 was treated with CoCl2 (Sigma-Aldrich, St. Louis, USA) to establish the cell hypoxia model as described previously [29 (link)]. AML12 cells were cultured in DMEM/F12 (Hyclone, Logan, USA) containing 10% fetal bovine serum (FBS), ITS liquid media supplement (Sigma-Aldrich, St. Louis, USA), and 40 ng/mL dexamethasone (Solarbio, Beijing, China) at 37°C with 5% CO2. The cells divided into four treatment groups. These were the control, ORY (240 μg/mL), CoCl2 (300 μM), and CoCl2+ORY (240 μg/mL ORY was added at the same time with CoCl2 (300 μM)) groups [30 (link)]. Levels of ROS in AML12 were determined by DCFH-DA ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer's instruction. The mouse hepatocyte cell line AML12 was treated with tunicamycin (TM) (Solarbio, Beijing, China) as described previously [31 (link)]. These were the control, ORY (240 μg/mL), TM (10 μg/mL), and TM+ORY. The cells were pretreated with γ-oryzanol for 12 h, followed by treatment with 10 μg/mL tunicamycin for an additional 24 h.
7
Melatonin Regulation of Cellular Stress
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Melatonin was purchased from Sigma (M5250, USA). The molecular weight of Melatonin was 232.28 and its purity was more than 98%. We weighed 4.65 mg Melatonin powder and dissolved it with 100 μL anhydrous ethanol to obtain a solution with a concentration of 200 mmol/L for subsequent experiments. Tunicamycin (TM) was purchased from Solarbio (T8480, China). The molecular weight of TM was 830.9. We weighed 1 mg TM powder, dissolved it with 48.1 μL DMSO to obtain a solution with a concentration of 25 mmol/L for subsequent experiments. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was purchased from Solarbio (C6700, China). The initial concentration of the product is 50 mmol/L, which can be diluted directly according to the need. CCK-8 detection reagent (BB-4202) and Annexin V-FITC Apoptosis Detection Kit (BB-4101-2) were purchased from BestBio (China).
8
Cytotoxicity Assays in Human Lung Cell Lines
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Human A549, H1299, H1688, H446 cells, and a human normal bronchial epithelial cell line (HBE) were purchased from Shanghai Cell Library of Chinese Academy of Science. MG132, Chloroquine (CQ), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), Thapsigargin (TG), CHX, and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Tunicamycin (TM) and actinomycin D were obtained from Solarbio. Sodium 4-phenylbutyrate (4-PBA) was from Abcam. N-acetyl-l -cysteine (NAC) was from Beyotime. Rapamycin (Rapa) and salubrinal were purchased from Calbiochem and Santa Cruz, respectively.
9
Culturing Normal Human Hepatocytes for Emodin Studies
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The normal human hepatocyte cell line (L02) was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The L02 cells were cultured in complete RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, 11011-861, EVERY GREEN) and 1% penicillin-streptomycin (10 kU/mL-10 mg/mL, FG101-01, Transgene, China) and maintained in an incubator with 5% CO2 at 37°C. The culture medium was renewed every two days, and cells were passaged at 80% confluence using 0.25% trypsin-EDTA. Emodin was purchased from EFEBIO Company (E054323, 98% (HPLC), China). (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO or MT, SML0737, ≥98% (HPLC)) and 2-aminoethyl diphenylborinate (2-APB, #9754, 97% (HPLC)) were from Sigma-Aldrich (China). N-Acetyl-L-cysteine was acquired from Beyotime Biotechnology (NAC, S0077, >99%, China). Tunicamycin (TM) and doxorubicin (DOX) were purchased from Solarbio Life Science (T8480 and D8740, China), and their purities were both more than 98%.
10
Exploring ER Stress Pathways in Cells
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Tunicamycin (TM) and sodium phenylbutyrate (4-PBA) were purchased from Solarbio (Beijing, China). Antibodies against p-PERK andp-IRE1α were from Affinity Biosciences (Zhenjiang, China); GRP78 and CD31, from Abcam (Cambridge, UK); and CHOP, PCNA, and GAPDH, from Proteintech group (Wuhan, China). The secondary antibodies (goat anti-rabbit and anti-mouse) were purchased from Beyotime (Shanghai, China). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA, USA).
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