Ouabain (Sigma-Aldrich; St. Louis, MO) and hydroxyurea (Merck, Kenilworth, NJ) were dissolved in water, Wortmannin (Sigma-Aldrich), PIK90 (Merck), LY294002 (TocrisBioscience, Bristol, UK), Rapamycin (Merck), AZD8055 (Santa Cruz Biotechnologies), and Withaferin A (TocrisBioscience) in DMSO, and solutions were further diluted in E3 embryo medium to concentrations of 3mM (Ouabain), 50mM (hydroxyurea), 1 µM (Wortmannin), 5 µM (PIK90), 25 µM (LY294002), 1.1 µM (Rapamycin), 30 µM (AZD8055), and 30 µM (WithaferinA). Embryos were incubated in inhibitor solution starting from 34 hpf and scored at 54 hpf.
Withaferin a
Manufactured by Bio-Techne
Sourced in Germany, United States, United Kingdom
Withaferin A is a natural compound isolated from the Withania somnifera plant. It is a laboratory reagent used for research purposes. Withaferin A has been studied for its potential biological activities, but its specific core function is not provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Aflibercept, by Regeneron Pharmaceuticals (2 mentions)
Sp600125, by Bio-Techne (2 mentions)
Recombinant human vegf 165 protein, by R&D Systems (2 mentions)
Sb202190, by Bio-Techne (2 mentions)
Sb259063, by Bio-Techne (2 mentions)
Mannitol, by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
Ro5 3335, by Merck Group (2 mentions)
L glucose, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Anisomycin, by Santa Cruz Biotechnology (1 mentions)
Interleukin 6 il 6, by Thermo Fisher Scientific (1 mentions)
Sr11302, by R&D Systems (1 mentions)
Ruboxistaurin, by Selleck Chemicals (1 mentions)
Calphostin c, by Bio-Techne (1 mentions)
Penicillin streptomycin, by Kerafast (1 mentions)
G 6976, by Bio-Techne (1 mentions)
Sotrastaurin, by Selleck Chemicals (1 mentions)
Formic acid, by Carl Roth (1 mentions)
6 protocols using withaferin a
1
Modulation of Zebrafish Development
2
Inhibitors and Activators of Key Signaling Pathways
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Tumor Necrosis Factor‐alpha (TNF‐α), Transforming Growth Factor beta 1 (TGFβ1), Transforming Growth Factor beta 2 (TGFβ2) and Interleukin 6 (IL‐6) were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant human VEGF 165 protein was purchased from R&D Systems (Minneapolis, MN, USA). Aflibercept (Eylea) was purchased from Regeneron Pharmaceuticals (Tarrytown, NY, USA). d ‐glucose, mannitol, L‐glucose, and RUNX1 inhibitor Ro5‐3335 were purchased from Millipore‐Sigma (Burlington, MA, USA). Small‐molecule inhibitors and activators purchased from commercial sources included TNF‐α‐TNFR1 binding inhibitor CAY10500 and JNK activator anisomycin, (Santa Cruz Biotechnology, Dallas, TX, USA); NF‐κB inhibitors Caffeic acid phenethyl ester (CAPE) and Honokiol; dual NF‐κB and JNK inhibitor Withaferin A, JNK inhibitors SP600125 and TCS JNK 6o; p38/MAPK inhibitors SB259063 and SB202190 (Tocris Bioscience, Bristol, UK); and AP‐1 inhibitor, SR11302 (R&D Systems). The CBFβ‐RUNX1 protein–protein interaction inhibitor, AI‐14‐91, and an inactive control compound of similar chemical structure, AI‐4‐88, were synthesized as described previously.36
3
Osteoblast and Osteocyte Cell Culture
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Cell culture and experiments with UMR106 rat osteoblast-like cells (purchased from ATCC, Manassas, VA, USA) were conducted as described before [39 (link)]. Briefly, cells were cultured in DMEM high-glucose medium containing 10% FBS and penicillin-streptomycin at 37°C and 5% CO2.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
4
Osteoblastic Cell Response to Acidic Conditions
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Cell culture experiments were conducted with UMR106 rat osteoblastic osteosarcoma cells (CRL-1661; ATCC, Manassas, VA, USA) cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Life Technologies) under standard culture conditions. Cells were pretreated with 10 nM 1,25(OH)2D3 (Tocris, Bristol, UK) for 24 h (6-well format; 2 × 105 cells/well). Twenty-four hours later, they were treated with the indicated concentration of L-lactic acid or Sodium (Na+)-L -lactate (sodium chloride as vehicle control; Sigma–Aldrich, Schnelldorf, Germany; 24 h), nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB) inhibitors withaferin A (Tocris; 500 nM, 24 h) or wogonin (Sigma; 100 µM, 24 h), or with vehicle only. withaferin A and wogonin are potent inhibitors of NFκB signaling [55 (link)–57 (link)] that is a major enhancer of Fgf23 gene expression [58 (link)]. In further series of experiments, UMR106 cells were treated with 22.8 mM formic acid (Carl Roth, Karlsruhe, Germany), 10 mM acetic acid (Carl Roth), or water for 24 h and, pH of supernatants was measured.
5
Rat Osteoblast Cell Culture and Treatments
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Rat osteoblast-like UMR106 cells (CRL-1661; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM glucose and 1 mM pyruvate (Gibco, Life Technologies, Thermo Scientific, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies) at 5% CO2 and 37 °C. Serum depletion was accomplished for 24 h or 48 h by incubating the cells in culture medium with 1% or 0% FBS and additional 10 nM 1,25(OH)2D3 (Tocris, Bioscience, Bristol, UK) to enhance Fgf23 expression [40 (link)]. Cells were seeded into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) for 24 h. Subsequently, cisplatin, PAC-1 or doxorubicin (all from Tocris Bioscience) were added at the indicated concentrations for 24 or 48 h or the FBS concentration was reduced as described above. Il-6 signaling was blocked through gp130 inhibitor SC144 (1 µM, Tocris Bioscience). NFκB inhibitors withaferin A (Tocris Bioscience) and wogonin (Merck, Darmstadt, Germany) were used at concentration of 500 nM and 100 µM, respectively, where indicated.
To study cell proliferation, cells were trypsinized after 24 h or 48 h, respectively, and counted on a Neubauer hemocytometer.
To study cell proliferation, cells were trypsinized after 24 h or 48 h, respectively, and counted on a Neubauer hemocytometer.
6
Modulation of Signaling Pathways in Cell Culture
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