Animals were incubated in 30 ml of sea water containing 1μM Sphingosine-Kinase-Inhibitor (A) SKI-I (Abcam), 14μM Sphingosine-Kinase-Inhibitor (B) CAS 1177741-83-1 (Millipore 567731), or 10 μM S1PR1 antagonist W146 (Tocris). Seawater containing drugs was replaced and fresh food (algae) was added daily. Controls were incubated in sea water without inhibitors with vehicle ethanol 0.1% or DMSO 0.001%. After 4 days, animals were fixed and analyzed by in situ hybridization as described above. For each treatment, two genetically identical colonies were treated simultaneously, and the experiment was performed three times. Buds containing vasa-positive cells and the number of vasa-positive cells in the vasculature were counted on each treated and untreated colony under an epifluoresence microscope. Data are expressed as averages from three experiments, and statistical analysis was performed using a paired, two-sided Student’s t-test.
Ski 1
Manufactured by Abcam
Sourced in United Kingdom
SKI-I is a small molecule compound that functions as an inhibitor of the proto-oncogene serine/threonine-protein kinase (PBK/TOPK). It is commonly used in cell-based and biochemical assays to study the role of PBK/TOPK in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Cas 1177741 83 1, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Tgf β1 antibody, by R&D Systems (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Saracatinib, by Santa Cruz Biotechnology (1 mentions)
Countess cell counter, by Thermo Fisher Scientific (1 mentions)
Enspire 2300, by PerkinElmer (1 mentions)
A419259, by Bio-Techne (1 mentions)
Cytotoxicity detection kitplus, by Roche (1 mentions)
Wst 8, by Roche (1 mentions)
Azd8055, by Merck Group (1 mentions)
Epz015666, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
6 protocols using ski 1
1
Investigating Sphingosine Kinase Inhibitors in Cnidarian Stem Cell Regulation
2
Characterizing Melanoma-Macrophage Interactions
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Melanoma cell lines were obtained from ATCC and grown as monolayers in RPMI or DMEM media supplemented with 10% heat-inactivated fetal calf serum (FCS) in the presence of 5% CO2 in a humidified atmosphere at 37°C. To guarantee cell line authenticity, B16F10 and COLO829 cell lines were used for a limited number of passages and routinely tested for the expression of melanocyte-lineage proteins such as MelanA/MART1. Human monocytic THP-1 cells obtained from Dr. A. Coste (University of Toulouse, France) were cultured in RPMI containing 10% FCS and 0.02 mM β-mercaptoethanol. THP-1 cells were differentiated into macrophages by stimulation with 20 ng/ml PMA (Sigma) for 24 hours; then cells were cultured for an additional 24 hours without PMA.
Conditioned media (CM) were produced by culturing melanoma cells in serum-free RPMI for 48 hours. In some experiments, melanoma cells were treated with 3 μM sphingosine kinase inhibitor SKI-I (5-(2-Naphthalenyl)-1H-pyrazole-3-carboxylic acid 2-[(2-hydroxy-1-naphthalenyl)methylene]hydrazide; Abcam). At the end of the culture period, the CM were collected, centrifuged for 5 min at 1500 rpm and filtered. Macrophages were then treated with the CM alone or in the presence of 5 μM S1P, 50 ng/ml recombinant TGF-β1 (Ebioscience SAS, Paris, France) or 1 μg/ml TGF-β1 antibody (Clone #1D11, R&D systems, Lille, France) for 48 hours.
Conditioned media (CM) were produced by culturing melanoma cells in serum-free RPMI for 48 hours. In some experiments, melanoma cells were treated with 3 μM sphingosine kinase inhibitor SKI-I (5-(2-Naphthalenyl)-1H-pyrazole-3-carboxylic acid 2-[(2-hydroxy-1-naphthalenyl)methylene]hydrazide; Abcam). At the end of the culture period, the CM were collected, centrifuged for 5 min at 1500 rpm and filtered. Macrophages were then treated with the CM alone or in the presence of 5 μM S1P, 50 ng/ml recombinant TGF-β1 (Ebioscience SAS, Paris, France) or 1 μg/ml TGF-β1 antibody (Clone #1D11, R&D systems, Lille, France) for 48 hours.
3
Influence of Sphingolipid Signaling on Vasa Expression
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Animals were incubated in 30 ml of sea water containing 1 μM Sphingosine Kinase Inhibitor (A) SKI-I (Abcam), 14 μM Sphingosine Kinase Inhibitor (B) CAS 1177741-83-1 (Millipore 567731) or 10 μM S1PR1 antagonist W146 (Tocris). Sea water containing drugs was replaced and fresh food (algae) was added daily. Controls were incubated in sea water without inhibitors with vehicle 0.1% ethanol or 0.001% dimethylsulphoxide. After 4 days, animals were fixed and analysed by in situ hybridization as described above. For each treatment, two genetically identical colonies were treated simultaneously, and the experiment was performed three times. Buds containing vasa-positive cells and the number of vasa-positive cells in the vasculature were counted on each treated and untreated colony under an epifluoresence microscope. Data are expressed as averages from three experiments, and statistical analysis was performed using a paired, two-sided Student's t-test.
4
Cell Viability and Cytotoxicity Assays
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Cell viability assays were performed using WST-8 (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions18 (link). Cells were cultured in 24-well plates. Twenty-four hours after treatment with the indicated concentrations of A419259 (Tocris Bioscience), SKI-1 (Abcam, Cambridge, UK), PP2 (Cayman Chemical, MI, USA), saracatinib (Santa Cruz) or bosutinib (abcam), cells were incubated with the WST-8 substrate for 2–3 h, after which the absorbance of the wells was measured at 450 nm using a plate reader (2300 EnSpireTM, PerkinElmer, Yokohama, Japan). The number of live cells was determined using a Countess cell counter (Invitrogen) after cultures were stained with trypan blue. Toxicity assays were performed using the Cytotoxicity Detection Kit PLUS (Roche Diagnostics, Indianapolis, IN). Twenty-four hours after treating the cells, 100 μL of the medium were removed from the plate and used in the assay. The medium was incubated with the substrate for 15 min and spectrophotometrically assayed at 490 nm using a plate reader. To activate the Src vector, we used MLR1023 (#4582) (Tocris Bioscience, Bristol, UK).
5
Cell culture and drug treatment
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RPE1 and MCF7 (American Type Culture Collection) cells were grown in DMEM supplemented with 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin. HT29 cells were growth in McCoy media supplemented with 10% FBS and Pen/Strep as above. Aneuploid cells RPE1 with trisomy 12 and trisomy 5 (Ts12 Ts5) and RPE1 hTert cells were kindly provided by the Storchova laboratory (Stingele et al, 2012 (link)). Most drugs used in this study were synthesized by Syncom B.V., except for AZD8055 (Sigma-Aldrich), EPZ015666 (Sigma-Aldrich) and SKI-1 (Abcam). All drugs were dissolved in DMSO (Sigma-Aldrich) and diluted in tissue culture medium as indicated. Used drug concentrations were titrated before the actual screen, starting from an initial drug concentration of 10 μM. If the initial drug concentration of 10 μM was (near-)toxic to wild-type RPE1 cells, the cells were next exposed to 1 μM, 0.1 μM, 10 nM, or 1 nM of the compound, until a concentration was found that was no longer toxic (see Supplemental Data 3 for all initial drug titration growth curves).
6
Combination Inhibition of STAT3 and SRC in NSCLC
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Cell lines and culture. A549 and H1975 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VI, USA). PC9 was obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). All cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS) in a humidified 5% CO 2 incubator at 37˚C. These cells are histologically adenocarcinomas, and A549 has the wild-type (WT) EGFR and KRAS mutations (codon 12), PC-9 has the EGFR mutation (exon 19 del), and H1975 has the EGFR mutation (L858R and T790M). A549 and H1975 cells have been reported to be EGFR-TKI resistant, whereas PC9 is EGFR-TKI sensitive. To test whether there was concomitant inhibition of STAT3 and SRC, cells were treated with combinations of different concentrations of a STAT3 inhibitor (S3I-201; Santa Cruz Biotechnology, Dallas, TX, USA) and SRC inhibitor (SKI-1; Abcam, Cambridge, UK). Drug concentrations of S3I-201 were adjusted to 0, 100, 200 and 400 µM and those for SKI-1 were adjusted to 0, 2.5, 5 and 10 µM.
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