Nsc95397

Manufactured by Merck Group

Sourced in United States, Germany

NSC95397 is a chemical compound used in research laboratories. It functions as a protein kinase inhibitor, which means it can interfere with the activity of certain enzymes involved in cellular signaling pathways. This compound is often utilized in scientific studies to investigate various biological processes.

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Lab products found in correlation

Paclitaxel, by Merck Group (2 mentions) Paclitaxel, by Qilu Pharmaceutical (1 mentions) Cell counting kit 8 cck 8 cell assay kits, by BestBio (1 mentions) Putrescine, by Merck Group (1 mentions) Selenium, by Merck Group (1 mentions) Anti ngf, by Merck Group (1 mentions) Anti phospho rb antibody, by Cell Signaling Technology (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Poly d lysine, by Merck Group (1 mentions) Dmem cell culture media, by Thermo Fisher Scientific (1 mentions) Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Real time pcr kit, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by GE Healthcare (1 mentions) Ecl reagent, by GE Healthcare (1 mentions) Nerve growth factor (ngf), by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Neurobasal, by Thermo Fisher Scientific (1 mentions)

11 protocols using nsc95397

Measuring Chemo-Resistance with CCK-8 Assay

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Cell viability was measured using Cell Counting Kit-8 (CCK-8) cell assay kits (Bestbio, Shanghai, China) according to the manufacturer's protocol. When MCS was established, cells were cultured in fresh media with DDP (in the final concentration of 30 nM or 90 nM) (Qilu Pharmaceutical, Jinan, Shandong, China) or paclitaxel (in the final concentration of 10 nM or 20 nM) (Qilu Pharmaceutical, Jinan, Shandong, China) for 48h. Cell viabilities were calculated based upon the OD values at 450nm. To measure the effect of NSC95397 (Millipore, Darmstadt, Germany) on chemo-resistance, we pre-treated cells with 32 nM NSC95397 for 48h before cultured in media with DDP or paclitaxel.

Sun Y., Li S., Yang L., Zhang D., Zhao Z., Gao J, & Liu L. (2019). CDC25A Facilitates Chemo-resistance in Ovarian Cancer Multicellular Spheroids by Promoting E-cadherin Expression and Arresting Cell Cycles. Journal of Cancer, 10(13), 2874-2884.

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In Vitro Neurodegeneration Assay

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Insulin, progesterone, putrescine, selenium, transferrin, anti-NGF, NGF, poly-d-lysine, NSC663284 and NSC95397 were purchased from Sigma (St Louis, MO, USA). Cell culture media DMEM, DMEM-F12, RPMI-1640, Neurobasal, B27, antibiotics, Lipofectamine 2000, Alexa Fluor, cDNA synthesis kit, real-time PCR kit and serum were purchased from Invitrogen (Life technologies, Grand Island, NY, USA). Cell culture dishes, plates and flasks were purchased from BD Falcon, Corning (Corning, NY, USA). Aβ_(1–42) and Aβ_(42-1) were purchased from American Peptide (Sunnyvale, CA, USA). ECL reagent and PVDF membrane were purchased from GE Healthcare (Buckinghamshire, UK). The PCR kits were purchased from Takara (Shiga, Japan), Fermentas (Waltham, MA, USA). Anti-Cdc25A, anti-Actin and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-Rb antibody was purchased from Cell Signaling Technologies (Denver, MA, USA). AKT inhibitor II, JNK inhibitor II and U0126 were purchased from Calbiochem (Darmstadt, Germany). Primers were purchased from IDT DNA (Gurgaon, Haryana, India). Brain tissues of AβPPswe-PS1de9 mice and control littermates were a kind gift from Dr. Anant B. Patel (Council of Scientific and Industrial Research-Centre for Cellular and Molecular Biology (CSIR- CCMB)), Hyderabad, India.

Chatterjee N., Sanphui P., Kemeny S., Greene L.A, & Biswas S.C. (2016). Role and regulation of Cdc25A phosphatase in neuron death induced by NGF deprivation or β-amyloid. Cell Death Discovery, 2, 16083-.

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Investigating NF-κB and AP-1 Signaling in IL-1β Response

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Cells were treated with two NF-κB inhibitors, TPCA1 (5 μM, Sigma-Aldrich, #T1452), and BOT-64 (5 μM, Santa Cruz Biotechnology, #sc-222062), and two AP-1 inhibitors, T-5224 (50 μM, GLPBIO, Montclair, CA, USA, #GC16165) and SR11302 (50 μM, Santa Cruz Biotechnology, #sc-204295), for 4 h and then stimulated with 50 ng/mL recombinant IL-1β for 2 h. To block CtBP2-mediated signaling, cells were treated with two CtBP inhibitors, MTOB (5 μM, MedKoo Biosciences, Morrisville, NC, USA, #561414) and NSC95397 (5 μM, Sigma-Aldrich, #N1786). In addition, cells were also only treated with different concentrations (0, 5, 50 and 500 ng/mL) of IL-1β. The resulting cells were applied to RNA or protein isolation to detect gene expression or protein level changes.

Zhou P., Wan X., Zou Y., Chen Z, & Zhong A. (2020). Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure. International Journal of Biological Sciences, 16(2), 204-215.

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CtBP1-Mediated Enzymatic Reduction Assays

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The reduction of 4-methylthio-2-oxobutanoic acid (MTOB) (Sigma-Aldrich) and NSC95397 (Sigma-Aldrich) by CtBP1 was analyzed spectrophotometrically by monitoring the disappearance of NADH absorbance at 340 nm using the Spectramax Plus absorbance cuvette reader. Assays were conducted in reaction mixtures (100 μL) containing PBS, 2 μM CtBP1, and 100 μM NADH after a 2-minute incubation at 30 °C.
The enzymatic assay of lactate dehydrogenase was carried out with 5 nM of lactate dehydrogenase (SigmaAldrich) and incubated with 5 μM pyruvate or varying concentrations of NSC93397 in PBS with 100 μM NADH for 1 min at 30 °C. The consumption of NADH was then monitored spectrophotometrically at 340 nm.

Blevins M.A., Kouznetsova J., Krueger A.B., King R., Griner L.M., Hu X., Southall N., Marugan J.J., Zhang Q., Ferrer M, & Zhao R. (2014). Small molecule, NSC95397, inhibits the CtBP1-protein partner interaction and CtBP1-mediated transcriptional repression. Journal of biomolecular screening, 20(5), 663-672.

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Exosome-Mediated Immune Modulation in ALI

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The same amounts of exosomes isolated from healthy control-sourced and ALI-sourced monocytes were added into a 12-well plate to coculture with U937 cells. After incubation at 37°C for 24 h, the adherent cells were collected for RNA and protein isolation. Similarly, the same amounts of two sourced exosomes were also injected into 6-week-old C57BL/6 mice (n = 20 for the control exosome group, and n = 60 for the ALI-sourced exosome group). Four hours after the injection, mice in the ALI-sourced exosome group were further randomly divided into three groups. The first group of mice was injected with PBS, the second group was injected with NSC95397 (5 mg/kg) (N1786; Sigma-Aldrich), and the third group was injected with MTOB (5 mg/kg) (82890; Sigma-Aldrich). The treated mice were left for 24 h, followed by blood and lung tissue collection to evaluate their inflammatory response.

Chen Z., Dong W.H., Qiu Z.M, & Li Q.G. (2020). The Monocyte-Derived Exosomal CLMAT3 Activates the CtBP2-p300-NF-κB Transcriptional Complex to Induce Proinflammatory Cytokines in ALI. Molecular Therapy. Nucleic Acids, 21, 1100-1110.

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Immunochemical Detection of Phosphorylated NIP30

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Anti-Flag-mouse and β-actin-mouse were obtained from MBL, anti-HA-mouse, anti-γH2AX-rabbit and anti-REGγ-rabbit were obtained from Abmart, anti-NIP30-rabbit, anti-p21-rabbit, and anti-CDC25A-rabbit were obtained from Proteintech. The rabbit antiserum against NIP30 phosphorylated at Ser-228 and Ser 230 were raised by HuaBio Ltd (China) using the synthetic peptide (CSDESS (pS) DSEGTIN / CSDESSSD (pS) EGTIN) as antigen. The antisera were pre-cleaned by affinity chromatography using the corresponding non-phosphorylated peptide (CSDESSSDSEGTIN) coupled to SulfoLink Resin (Thermo Fishfer Scientific), and then purified by affinity chromatography with a phosphor-peptide. NSC95397, a CDC25A inhibitor, was purchased from Sigma. Purified CDC25A (ab90763) protein was purchased from Abcam.

Gao X., Wang Q., Wang Y., Liu J., Liu S., Liu J., Zhou X., Zhou L., Chen H., Pan L., Chen J., Wang D., Zhang Q., Shen S., Xiao Y., Wu Z., Cheng Y., Chen G., Krubra S., Qin J., Huang L., Zhang P., Wang C., Moses R.E., Lonard D.M., Malley B.W., Fares F., Zhang B., Li X., Li L, & Xiao J. (2020). The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells. Nature Communications, 11, 3904.

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Chemical Toxicity Evaluation on Stem Cells

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We conducted detailed studies of chemical toxicity on hiPSCs, hMSCs, and their differentiated progenies at various stages of differentiation (Figure S1). Cells were treated with different chemicals in 96-well plates and cell viability was measured with the CCK-8 assay (Sigma-Aldrich, Cat. No. 96992). For all chemical toxicity assays, 6,000 hMSCs in 100 μL medium were seeded per well in a 96-well plate, and treated for 48 hours at 37°C/5% CO₂ before cell viability readout on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). A total of 14 different concentrations ranged from 0.0029 to 223 μM were used for each chemical in triplicates. The four chemicals, AC-93253, idarubicin, NSC 95397, and cantharidin, were purchased from Sigma, and dissolved in 100% DMSO. For chemical toxicity test on hMSCs, the final DMSO concentration in the media was 0.5%. For hiPSCs and hepatic differentiation Phase I cells, the final DMSO concentration was 0.1%. For hepatic differentiation Phase II and Phase III chemical toxicity tests, the final DMSO concentration for NSC 95397 was 0.3%, while 0.1% for the other 3 chemicals. Each chemical toxicity tests were repeated at least three times. The cell viabilities were plotted versus concentrations for each chemical. GraphPad Prism 6 was used to fit the data to a 3-parameter dose-inhibition curve to obtain the IC₅₀ values.

Han Y., Zhao J., Huang R., Xia M, & Wang D. (2017). Omics-based platform for studying chemical toxicity using stem cells. Journal of proteome research, 17(1), 579-589.

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Peptide Synthesis and Delivery

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Preparation of the PLDLS-containing Pep1-E1A and the PLDLS-to-PLDEL mutant (Pep1-E1A^Mut) peptides were as previously described 41 (link). Peptides (6 mg/mL) and MTOB sodium salt (Sigma, K6000; 425 mg/mL) were dissolved in water. NSC95397 (Sigma, N1786; 5 mg/mL) was dissolved in DMSO. The stock solutions were diluted in PBS solution (vehicle) to desired final concentrations freshly before use. Pep1-E1A was delivered at 2 mg/kg dosage, 4-methylthio-2-oxobutyric acid (MTOB) sodium salt at 860 mg/kg and NSC95397 at 0.75 or 1.5 mg/kg with intraperitoneal (i.p.) injection.

Li H., Zhang C., Yang C., Blevins M., Norris D., Zhao R, & Huang M. (2020). C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment. International Journal of Biological Sciences, 16(7), 1107-1120.

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High-Throughput Screening of Mitotic Regulators

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The drugs used for screening in this study are listed in Supplementary Table 1.
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.

Jemaà M., Abdallah S., Lledo G., Perrot G., Lesluyes T., Teyssier C., Roux P., van Dijk J., Chibon F., Abrieu A, & Morin N. (2016). Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells. Oncotarget, 8(10), 16669-16689.

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Cell Treatments with Small Molecules

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Cells at 80% confluency were washed twice with PBS buffer, followed by treatments with 2 µM NSM00158, 20 µM NSC95397 (Sigma-Aldrich, #N1786), or 1 µM RCM1 (Sigma-Aldrich, #SML2625). Cells were further incubated at 37°C for 6 h, and then were collected and used in experiments.

Chen X., Zhang Q., Dang X., Song T., Wang Y., Yu Z., Zhang S., Fan J., Cong F., Zhang W, & Duan N. (2021). Targeting the CtBP1-FOXM1 transcriptional complex with small molecules to overcome MDR1-mediated chemoresistance in osteosarcoma cancer stem cells. Journal of Cancer, 12(2), 482-497.

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