F-Actin ring formation assays are carried to detect the effect of RTA-408 on the generation of mature osteoclasts. The actin rings were detected using phalloidin with fluorescence microscopy after RTA-408 treatment (20 nM).
The protein level of STING was analyzed by western blotting on day 2 and 4 of osteoclastogenesis in the presence of RTA-408.
Omaveloxolone purchased from MedChemExpress. Usage Cited in:
Oxid Med Cell Longev. 2017;2017:7612182.
[Abstract]
Nuclear translocation of Nrf2 by RTA-408 is measured by immunohistochemistry staining.
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Description
Omaveloxolone (RTA 408) is an antioxidant inflammation modulator (AIM), which activates Nrf2 and suppresses nitric oxide (NO). Omaveloxolone attenuates osteoclastogenesis by inhibiting STING dependent NF-κb signaling.
IC50 & Target
Nrf2[1]
Cellular Effect
Cell Line
Type
Value
Description
References
HT-29
EC50
1.41 μM
Compound: 12; RTA-408
Anti-necroptotic activity in human HT-29 cells assessed as inhibition of TNFalpha/SM-164/Z-VAD-fmk (TSZ)-induced necroptosis by measuring increase in cell viability measured after 12 hrs by celltiter-glo luminescent cell viability assay
Anti-necroptotic activity in human HT-29 cells assessed as inhibition of TNFalpha/SM-164/Z-VAD-fmk (TSZ)-induced necroptosis by measuring increase in cell viability measured after 12 hrs by celltiter-glo luminescent cell viability assay
[PMID: 33248849]
L929
EC50
5.25 μM
Compound: 12; RTA-408
Anti-necroptotic activity in mouse L929 cells assessed as inhibition of TNFalpha/Z-VAD-fmk (TZ)-induced necroptosis by measuring increase in cell viability measured after 12 hrs by celltiter-glo luminescent cell viability assay
Anti-necroptotic activity in mouse L929 cells assessed as inhibition of TNFalpha/Z-VAD-fmk (TZ)-induced necroptosis by measuring increase in cell viability measured after 12 hrs by celltiter-glo luminescent cell viability assay
[PMID: 33248849]
In Vitro
To evaluate the anti-inflammatory activity of Omaveloxolone (RTA 408), RAW 264.7 mouse macrophage cells are treated with Omaveloxolone for two hours and then IFNγ is added to stimulate NO production and release into the media. Omaveloxolone dose-dependently reduces NO concentrations in the media with an IC50 value of 4.4±1.8 nM. The potency of Omaveloxolone in this assay is similar to that of Bardoxolone methyl, which has an IC50 value of 1.9±0.8 nM. Nrf2 activation is required for AIM-mediated NO suppression. A decrease in nitric oxide synthase 2 (Nos2) protein levels is observed in bardoxolone methyl-treated RAW 264.7 cells, which is attenuated when Nrf2 mRNA levels are reduced by siRNA. To evaluate the anticancer activity of Omaveloxolone, a panel of eight human cell lines derived from tumors of different origin are treated with Omaveloxolone and measured cell growth 72 hours later using the sulforhodamine B (SRB) assay. Omaveloxolone inhibits the growth of all tumor lines with an average GI50 value of 260±74 nM. To determine whether Omaveloxolone induces apoptosis, the panel of tumor cells are treated with Omaveloxolone and the caspase substrate, DEVD-AFC, for 24 hours. Omaveloxolone dose-dependently increases DEVD-AFC cleavage, indicating that Omaveloxolone treatment triggers caspase activation in cancer cells. Caspase-3 and caspase-9 cleavage is also observed by western blot at the same concentrations of Omaveloxolone that increases DEVD-AFC cleavage[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Omaveloxolone Related Antibodies
In Vivo
To determine whether Omaveloxolone (RTA-408) is an effective mitigator of hematopoietic acute radiation syndrome after bone marrow-lethal doses of total-body irradiation (TBI), mice are administered 3 daily injections of 17.5 mg/kg Omaveloxolone beginning 24 h after TBI. Teatment with Omaveloxolone results in the 35 day survival of 100% of 7 Gy (LD40/35) TBI mice (P<0.05) and 60% of 7.5 Gy (LD100/13) TBI mice (P<0.0001)[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Room temperature in continental US; may vary elsewhere.
Storage
Powder
-20°C
3 years
4°C
2 years
In solvent
-80°C
2 years
-20°C
1 year
Solvent & Solubility
In Vitro:
DMSO : 100 mg/mL (180.27 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
Preparing Stock Solutions
ConcentrationSolventMass
1 mg
5 mg
10 mg
1 mM
1.8027 mL
9.0137 mL
18.0274 mL
5 mM
0.3605 mL
1.8027 mL
3.6055 mL
10 mM
0.1803 mL
0.9014 mL
1.8027 mL
View the Complete Stock Solution Preparation Table
*Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles. Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.
For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day. The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Protocol 1
Add each solvent one by one: 10% DMSO 90% Corn Oil
Solubility: ≥ 2.5 mg/mL (4.51 mM); Clear solution
This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Taking 1 mL working solution as an example, add 100 μLDMSO stock solution (25.0 mg/mL) to 900 μLCorn oil, and mix evenly.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:
Dosage
mg/kg
Animal weight (per animal)
g
Dosing volume (per animal)
μL
Number of animals
Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO+
%
+
%
Tween-80
+
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO,
. All of co-solvents are available by MedChemExpress (MCE).
, Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration:
mg/mL
Method for preparing stock solution:
mg
drug dissolved in
μL
DMSO (Stock solution concentration: mg/mL).
The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take
μL DMSO stock solution, add
μL .
μL , mix evenly, next add
μL Tween 80, mix evenly, then add
μL Saline.
Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution
If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
[1]. Probst BL, et al. RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity. PLoS One. 2015 Apr 21;10(4):e0122942.
[Content Brief]
[2]. Peng Han, et al. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017.
[3]. Goldman DC, et al. The triterpenoid RTA 408 is a robust mitigator of hematopoietic acute radiation syndrome in mice. Radiat Res. 2015 Mar;183(3):338-44
[Content Brief]
Cell Assay
[1]
MEFs, PANC-1, A549, A375, A549/NF-κB-Luc and HeLa/NF-κB-Luc cells are cultured in Gibco high glucose DMEM with 10% FBS. G-361 cells are cultured in McCoy’s 5A medium with 10% FBS. All other cell lines are cultured in RPMI 1640 medium with 10% FBS. For growth inhibition assays, cells are plated in duplicate 96-well culture dishes at 3×103 cells per well. The following day, one plate is treated with Omaveloxolone (200, 400, 600, 800 and 1000 nM) and the other is immediately processed for the sulforhodamine B (SRB) assay (time 0). Cells in the Omaveloxolone-treated plate are processed for the SRB assay 72 hours after the start of treatment. Percentage of growth relative to vehicle-treated cells is calculated. Dose-response curves are plotted in GraphPad Prism and GI50 values are calculated. For cell counting experiments, MEFs are plated in 6-well culture dishes at 5×104 cells per well and treated with Omaveloxolone the following day. Following treatment, cells are counted using a Vi-CELL XR cell analyzer. For clonogenic assays, wild-type (1×103 cells per well) and Keap1-/- (0.5×103 cells per well) MEFs are seeded in 6-well dishes. Six hours later, MEFs are treated with Omaveloxolone. After seven days, colonies are fixed with a 1:7 solution of acetic acid:MeOH and stained with 0.5% crystal violet in MeOH. Colonies consisting of ≥50 cells are counted[1].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Animal Administration
[2]
Mice[2]
For radiation survival experiments, wild-type C57Bl/6 CD45.2 mice (6-8 weeks old) are used. Congenic wild-type C57Bl/6 CD45.1 and C57Bl/6 CD45.1/CD45.2 hybrid host mice are used as recipients in transplantation experiments. Omaveloxolone stock solutions for vehicle control (DMSO) are prepared within 1 h before injection. Omaveloxolone (17.5 mg/kg) or DMSO is administered intraperitoneally at 24, 48 and 72 h after irradiation. Whole-body irradiation (7-8 Gy) is performed using a 250-kVp X-ray machine with 50 cm source-to-skin distance and a 2 mm copper filter. The dose rate is approximately 1.4 Gy/min.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
References
[1]. Probst BL, et al. RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity. PLoS One. 2015 Apr 21;10(4):e0122942.
[Content Brief]
[2]. Peng Han, et al. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017.
[3]. Goldman DC, et al. The triterpenoid RTA 408 is a robust mitigator of hematopoietic acute radiation syndrome in mice. Radiat Res. 2015 Mar;183(3):338-44
[Content Brief]
[1]. Probst BL, et al. RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity. PLoS One. 2015 Apr 21;10(4):e0122942.
[2]. Peng Han, et al. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017.
[3]. Goldman DC, et al. The triterpenoid RTA 408 is a robust mitigator of hematopoietic acute radiation syndrome in mice. Radiat Res. 2015 Mar;183(3):338-44
Complete Stock Solution Preparation Table
*Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles. Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.
Optional Solvent
ConcentrationSolventMass
1 mg
5 mg
10 mg
25 mg
DMSO
1 mM
1.8027 mL
9.0137 mL
18.0274 mL
45.0686 mL
5 mM
0.3605 mL
1.8027 mL
3.6055 mL
9.0137 mL
10 mM
0.1803 mL
0.9014 mL
1.8027 mL
4.5069 mL
15 mM
0.1202 mL
0.6009 mL
1.2018 mL
3.0046 mL
20 mM
0.0901 mL
0.4507 mL
0.9014 mL
2.2534 mL
25 mM
0.0721 mL
0.3605 mL
0.7211 mL
1.8027 mL
30 mM
0.0601 mL
0.3005 mL
0.6009 mL
1.5023 mL
40 mM
0.0451 mL
0.2253 mL
0.4507 mL
1.1267 mL
50 mM
0.0361 mL
0.1803 mL
0.3605 mL
0.9014 mL
60 mM
0.0300 mL
0.1502 mL
0.3005 mL
0.7511 mL
80 mM
0.0225 mL
0.1127 mL
0.2253 mL
0.5634 mL
100 mM
0.0180 mL
0.0901 mL
0.1803 mL
0.4507 mL
Omaveloxolone Related Classifications
NF-κBImmunology/InflammationApoptosis
Keap1-Nrf2STINGApoptosis
Help & FAQs
Do most proteins show cross-species activity?
Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.
Keywords:
Omaveloxolone1474034-05-3RTA 408RTA408RTA-408Keap1-Nrf2STINGApoptosisStimulator of Interferon GenesTMEM173MITAERISMPYSInhibitorinhibitorinhibit
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