Oridonin (B20310) was purchased from Yuanye company (Shanghai, China. APR-246 was provided by MCE (USA). GSH and GSSG Assay Kit (S0053) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime. Annexin V-FITC Apoptosis Detection Kit (KGA106) was from KeyGEN. Lipofectamine 3000 Reagent (L3000008) was from Invitrogen. Anti-xCT (ab175186) and GAPDH Monoclonal Antibody (AC033) were supplied by Abcam and Abclonal, respectively. Goat Anti-Mouse IgG (H + L)/HRP antibody (bs-40296G-HRP) was from Bioss.
Oridonin
Manufactured by Yuanye Bio-Technology
Sourced in China
Oridonin is a natural bioactive compound extracted from the traditional Chinese medicinal plant Isodon rubescens. It is a white crystalline powder that serves as a key raw material for pharmaceutical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Recombinant rnase, by Takara Bio (2 mentions)
M mlv reverse transcriptase, by Takara Bio (2 mentions)
Sybr premix ex taqtm, by Takara Bio (2 mentions)
Trizol, by Roche (2 mentions)
Gapdh monoclonal antibody, by Abcam (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Gsh and gssg assay kit, by Beyotime (1 mentions)
Lps from escherichia coli 055 b5, by Merck Group (1 mentions)
Sybr green plus reagent kit, by Roche (1 mentions)
Baicalein, by Yuanye Bio-Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Dihydroartemisinin, by Yuanye Bio-Technology (1 mentions)
Andrographolide, by Yuanye Bio-Technology (1 mentions)
Sulforaphane, by Yuanye Bio-Technology (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
Shikonin, by Yuanye Bio-Technology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
12 protocols using oridonin
1
Oridonin and APR-246 Cytotoxicity Assay
2
Oridonin HPLC Purity Determination
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Oridonin (HPLC ≥ 98 %) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). The purity of Ori was determined by HPLC. The assay was performed on an EChrom2000 DAD Data System (Elite, Dalian, China). Chromatography was performed using a Hyper ODS-2 C18 column (5μm, 250×4.6 mm, Dikma Technology, California, USA). Elution was performed with acetonitrile/water (30:70), and the flow rate was 1.0 mL/min with DAD detection at 242 nm (Figure 1B ). LPS (from Escherichia coli 055: B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The indicated primary antibodies and actin were obtained from Cell Signalling Technology (Beverly, MA, USA). qPCR was carried out using the SYBR Green Plus Reagent kit (Roche Applied Science, Mannheim, Germany). All of the other chemicals and reagents were of the highest commercial grade available.
3
Inhibiting c-Myc Signaling Pathways
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c-Myc inhibitors (10,058-F4 and 10,074-G5) were obtained from Selleckchem (Houston, TX, USA). Artemisinin (ARTS), Dihydroartemisinin (DHA), Shikonin, Oridonin, Andrographolide, Baicalein, Sulforaphane and Luteolin (purity ≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). RPMI-1640, Dulbecco’s Modified Eagle’s medium (DMEM), Opti-MEM and fetal bovine serum (FBS) were purchased from Gibco/BRL (Grand Island, NY, USA). Sulforhodamine B (SRB) and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Homo c-Myc plasmid was obtained from Gene Pharma (Gene Pharma Co., Ltd., China). Primary antibodies against c-Myc and PD-L1 were purchased from Cell Signaling Technology (Beverly, MA, USA); β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
4
Aeromonas hydrophila Antibacterial Assays
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Aeromonas hydrophila AS 1.1801 was bought from the China Center for Type Culture Collection (CCTCC) and stored at −80 °C in nutrient broth (NB, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) with 20% glycerol. The frozen strains were reactivated via inoculation in nutrient agar (NA, Qingdao Hope Bio-Technology Co., Ltd.) and incubated at 28 °C for 24 h. The isolated colonies were inoculated into 50 mL of NB and incubated under stirring at 180 rpm on a constant temperature shaker (Boxun, Shanghai, China) at 28 °C for 20 h. These bacterial inocula cultures (~1 × 108 CFU/mL, 0.5 McFarland turbidity) were used for the antibacterial activity assays. Oridonin (˃98% HPLC purity; CAS no.28957-04-2) was bought from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and dissolved in methanol to obtain a stock solution.
5
Oridonin and Ferrostatin-1 Protocols
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Oridonin (HPLC ≥98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (#B20310), then the drug was dissolved in DMSO and stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) was purchased from MedChemExpress (#HY-100579, MCE), and stored in − 20 °C for usage. All primary antibody used in the experiments included Actin (1:10000, #60008–1-Ig, Proteintech), Bcl-2 (1:1000, #61193, Proteintech), BAX (1:5000, #50599–2-Ig, Proteintech), cl-caspase3 (1:10000, #19677–1-AP, Proteintech), Tublin (1:1000, #66031–1-Ig, Proteintech), GAPDH (1:2000, #CL594–60004, Proteintech), GPX4 (1:5000, #67763–1-IG, Proteintech), SLC7A11 (1:1000, #26864–1-AP, Proteintech), FTH1 (1:5000, #11682–1-AP, Proteintech), and ACSL4 (1:10000, #81196–1-RR, Proteintech), they were stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) (#HY-100579, MCE), an effective ferroptosis inhibitor [32 (link)], was dissolved in dimethylsulfoxide (DMSO) to create stock solutions and stored at − 20 °C.
6
Oridonin Cytotoxicity Evaluation in HepG2 Cells
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Oridonin (No: 1126YA13, purity more than 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., in China. A 100 g/L stock solution of Oridonin was diluted in 100% dimethyl sulfoxide (DMSO) and stored at 4 °C in darkness. A serial dilution was made in 100% DMSO that was 1000 times more concentrated to allow for a 1:1000 dilution with EM to create a serial dilution with a final DMSO concentration of 0.1%. HepG2-Luciferase cells were purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. Cell Tracker CM-Dil (No: 1,583,101) was purchased from Shanghai Qianchen Biological Technology Co., Ltd. RPMI1640 culture solution (No: 11,875,093) was purchased from Invitrogen. Annexin V-FITC (No: 556,547) and Matrigel (No: 354,234) were purchased from BD Pharmingen. Calcein-AM (No: 56,496), MTT (No: M5655), penicillin, streptomycin, DMSO, and tricaine methanesulfonate (MS-222) were obtained from Sigma. Bevacizumab (Avastin®) and Trizol (No: 11,667,165,001) were purchased from Roche. Anti-CD31, claudin-1 (No: 81,796), claudin-4 (No: 37,643), and claudin-7 (No: 17,670) were purchased from Sigma. Recombinant RNase (No: 2313A), Reverse TranScriptase M-MLV (No: 2641A), and SYBR Premix Ex TaqTM (No: RR420) were purchased from Takara. The other chemicals used in this study were of analytical grade.
7
Immunochemical Analysis of DNA Damage
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The RAD51 (ab133534) and γH2AX (ab81299) antibodies were purchased from Abcam. Glyceraldehyde-3-phosphate dehydrogenase (BBI D190636) antibody was purchased from Sangon. Cisplatin was purchased from Sigma, Oridonin was purchased from Shanghai Yuanye Biotechnology.
8
Cultivation and Characterization of Burkholderia Strains
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All the strains used in this study are listed in Table 1 . The B. cepacia complex, B. cenocepacia H111, rpfFBC mutant, cepI mutant, and Escherichia coli strains were grown at 37°C in LB medium (5 g yeast extract, 10 g tryptone, and 10 g/liter NaCl; solid medium also contained 15 g/liter agar). The following antibiotics were used to supplement the media when necessary: 100 μg/mL ampicillin, 100 μg/mL kanamycin, and 20 μg/mL tetracycline. The chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl β-d -galactopyranoside) was used at 40 μg/mL. Oridonin (Yuanye Bio-Technology, Shanghai, China; high-pressure liquid chromatography [HPLC] ≥ 99%) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mM, and this solution was added to the medium in the experiments. In the protease activity experiment, NYG medium (3 g yeast extract, 5 g peptone, and 20 g/liter glycerin) was used to culture B. cenocepacia H111. In the biofilm formation assay, minimal medium (2 g glycerin, 2 g mannitol, 10.5 g K2HPO4, 4.5 g KH2PO4, 2 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 0.005 g FeSO4, 0.01 g CaCl2, and 0.002 g/liter MnCl2) was used to culture B. cenocepacia H111. Bacterial growth was monitored spectrophotometrically by measuring the optical density at 600 nm.
9
Oridonin Preparation and Characterization
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Oridonin (No: 1126YA13, purity more than 98%) was purchased from Shanghai Yuanye Bio-Technology Co. Ltd., China. Oridonin stock solution (100 g/L) was diluted in 100% dimethyl sulfoxide (DMSO) and stored at 4 °C in the dark. Next, the solution was serially diluted in 100% DMSO that was 1000 times more concentrated to allow for a 1:1000 dilution with embryo medium (EM) (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3) (Westerfield 2000 ) to create a serial dilution with a final DMSO concentration of 1%. DMSO and tricaine methanesulfonate (MS-222) were obtained from Sigma. Trizol (No: 11667165001) was purchased from Roche. Recombinant RNase (No: 2313 A), Reverse TranScriptase M-MLV (No: 2641 A) and SYBR Premix Ex TaqTM (No: RR420) were purchased from Takara. The other chemicals used in this study were of analytical grade.
10
Cytotoxicity of LNT and Oridonin on A549 Cells
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The MTT assays were performed as described previously (16 (link)). A549 cells (4x104 cells/well) were seeded into 96-well plates in 100 µl supplemented media per a well the night before treatments. Different concentration of LNT (Shanghai Yuanye Biological Technology Co., Ltd.) and oridonin (Shanghai Yuanye Biological Technology Co., Ltd.) were added to the plates and cultured for 72 h. Subsequently, the culture medium was discarded, and 100 µl fresh culture media containing 0.5 mg/ml MTT was added (Nanjing Aoduofuni Biology Technology, Co. Ltd.), and the plates were further incubated for 4 h. The solutions were discarded and 100 µl DMSO was added to dissolve the crystals, Finally, the optical density of each well was measured at a wavelength of 540 nm using a microplate reader (Imar; Bio-Rad Laboratories, Inc.) and the ratio of suppression of viability induced by the different treatments was calculated. Based on preliminary results, 300 µg/ml LNT was used for the subsequent experiments as the high concentration group (LNT-H), as it was the concentration that had the most potent effect on the viability of cancer cells, whilst having little effect on the viability of normal lung cells. As a comparison, a low concentration group (LNT-L) was also included, in which cells were treated with 100 µg/ml LNT.
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