Sbi 0206965

Manufactured by Merck Group

Sourced in United States

SBI-0206965 is a small molecule compound developed by Merck Group. It is a laboratory reagent intended for use in research applications. The core function of SBI-0206965 is to serve as a tool for investigating biological processes and pathways. Further details about its intended use or applications are not available.

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Lab products found in correlation

Chloroquine, by Merck Group (6 mentions) Bafilomycin a1, by Merck Group (5 mentions) Rapamycin, by Merck Group (4 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Tween 80, by Merck Group (2 mentions) Spautin 1, by Merck Group (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) N acetyl l cysteine, by Merck Group (2 mentions) Staurosporine, by Merck Group (2 mentions) Bix 01294, by Merck Group (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Bafilomycin a1, by Enzo Life Sciences (2 mentions) Compound c, by Merck Group (2 mentions) Thapsigargin, by Merck Group (2 mentions) Z vad fmk, by R&D Systems (2 mentions) Mcf 7 cell, by American Type Culture Collection (1 mentions) Beclin 1, by Abcam (1 mentions) β actin, by Proteintech (1 mentions) Sf3b3, by Abcam (1 mentions) Goat anti mouse igg, by Cell Signaling Technology (1 mentions)

28 protocols using sbi 0206965

Autophagy Modulation in MCF-7 Breast Cancer Cells

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MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and used to conduct functional assays. The cells were cultured in DMEM medium supplemented with 10% FBS, 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.03% l-glutamine and maintained at 37 °C with 5% CO₂ at a humidified atmosphere. SBI-0206965, 3-MA, bafilomycin A1 and chloroquine were purchased from Sigma–Aldrich [the detail information are as follows: SBI-0206965 (SML1540), 3-MA (189490), bafilomycin A1 (196000) and chloroquine (C6628)]. The primary antibodies used for Western blot are as follows: LC3 B (Abcam, ab192890), beclin-1 (Abcam, ab207612), SF3B3 (Abcam, ab96683), ULK1 (8054, CST, MA, USA), p-ULK1^Ser317 (12753, CST), p-ULK1^Ser555 (5869, CST), mATG13 (13273, CST), p-mATG13^Ser318 (PAB19948, Abnova, Taiwan), ATG101 (13492, CST), FIP200 (12436, CST), SQSTM1/P62 (5114, CST), ATG4B (13507, CST), ATG4C (5262, CST), ATG5 (12994, CST), ATG16L1 (8089, CST), SIRT3 (2627, CST), ac-MnSOD2 K68 (Abcam, ab137037), ac-MnSOD2 K122 (Abcam, ab214675), MMP-2(40994, CST), MMP-9 (13667, CST), E-cadherin (14472, CST), and β-actin (66009-1-Ig, Proteintech, IL, USA). In addition, the secondary infrared antibodies goat anti-rabbit IgG (Cell Signaling Technology #4410, 1:5000) and goat anti mouse IgG (Cell Signaling Technology #4414, 1:5000) were added.

Zhang S., Zhang J., An Y., Zeng X., Qin Z., Zhao Y., Xu H, & Liu B. (2020). Multi-omics approaches identify SF3B3 and SIRT3 as candidate autophagic regulators and druggable targets in invasive breast carcinoma. Acta Pharmaceutica Sinica. B, 11(5), 1227-1245.

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AML Mouse Model Treatment Protocol

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SBI-0206965 (Sigma-Aldrich) and MRT-68921 (Sigma-Aldrich) were dissolved in DMSO and distilled water, respectively, to prepare the 10 mg/ml stock solution. AML-bearing mice were intraperitoneally injected with a dose of 20 mg/kg SBI-0206965 diluted in PBS containing 5% polyethylene glycol 400 (Sigma-Aldrich) and 5% Tween 80 (Sigma-Aldrich) or MRT-68921 diluted in PBS according to the schedule (Fig. 5a).

Baba T., Yoshida T., Tanabe Y., Nishimura T., Morishita S., Gotoh N., Hirao A., Hanayama R, & Mukaida N. (2021). Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway. Cell Death & Disease, 12(4), 322.

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Preparation and Storage of Experimental Compounds

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AICAR (Toronto Chemical Research) was prepared at a concentration of 1.3 × 10⁻¹ M in distilled water and kept at − 20 °C until use. 2-deoxyglucose (Sigma) was prepared at a concentration of 10⁻¹ M in distilled water and used immediately. SBI-0206965 (Sigma-Aldrich), A-769662 (Tocris Bioscience) and the different modulator candidates were prepared in DMSO (Sigma). NA (Sigma Aldrich) was prepared in a saline-ascorbic solution (0.9% NaCl/0.01% ascorbic acid), Ach and L-NAME (Sigma Aldrich) were prepared in 0.9% NaCl solution. A stock solution (10⁻² M) was prepared for each of them and stored at − 20 °C until use (maximum 3 months).

Sanz-Gómez M., Aledavood E., Beroiz-Salaverri M., Lagartera L., Vega-Martín E., Gil-Ortega M., Cumella J., Pérez C., Luque F.J., Estarellas C., Fernández-Alfonso M.S, & Castro A. (2022). Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK–eNOS–NO pathway. Scientific Reports, 12, 4225.

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Autophagy Modulation in Cell Infection

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Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rapamycin, 3-methyladenine (3-MA), Mdivi-1, SBI-0206965 (ULK1 inhibitor), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were treated with various doses of the drugs prior to infection.

Oh S.J., Lim B.K., Yun J, & Shin O.S. (2021). CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways. Frontiers in Cellular and Infection Microbiology, 11, 704494.

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Primary Keratinocyte Isolation and Culture

Cited 1 time

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All experiments were conducted using primary keratinocytes. Cells were isolated from normal human skin by previously described methods (Lazarov et al., 2002 (link), Ridky et al., 2010 (link)). Primary cells were obtained through the SBDRC core from deidentified discarded material though an IRB approved protocol. Keratinocytes were cultured in a 1:1 mixture of Gibco Keratinocytes-SFM medium + L-glutamine + EGF + BPE and Gibco Cascade Biologics 154 medium with 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). Transduced keratinocytes were fully puromycin selected before the commencement of each experiment. Lys05 (Gift from R. Amavaradi) was used at 2 μM, Bafilomycin-A1 (Sigma, St. Louis, MO) was at 10 nM. SBI-0206965 and Spautin-1 (Sigma, St. Louis, MO) were used at 2 μM, DDC (Abcam, Cambridge, MA) was used at 20 μM, N-acetylcysteine (Sigma, St. Louis, MO) was used at 10mM, EUK134 (Sigma, St. Louis, MO) was used 50 μM at and TEMPOL (Sigma, St. Louis, MO) was used at 1 mM.

Monteleon C.L., Agnihotri T., Dahal A., Liu M., Rebecca V.W., Beatty G.L., Amavaradi R.K, & Ridky T.W. (2018). Lysosomes support the degradation, signaling, and mitochondrial metabolism necessary for human epidermal differentiation. The Journal of investigative dermatology, 138(9), 1945-1954.

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Transient Transfection and Starvation Assay

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Transient transfections were performed using GeneJuice transfection reagent (Merck-Millipore) for U2OS cells or polyethylenimine linear MW 25,000 (PEI) for HEK293 cells according to the manufacturers’ protocols. siRNA transfections were carried out using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer.
BafilomycinA1 (Santa Cruz Biotechnology) was used in a concentration of 200 nM.
For induction of starvation, Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich) was used.
The ULK1 inhibitor SBI-0206965 (Sigma-Aldrich) was used in a concentration of 5 µM.

Holdgaard S.G., Cianfanelli V., Pupo E., Lambrughi M., Lubas M., Nielsen J.C., Eibes S., Maiani E., Harder L.M., Wesch N., Foged M.M., Maeda K., Nazio F., de la Ballina L.R., Dötsch V., Brech A., Frankel L.B., Jäättelä M., Locatelli F., Barisic M., Andersen J.S., Bekker-Jensen S., Lund A.H., Rogov V.V., Papaleo E., Lanzetti L., De Zio D, & Cecconi F. (2019). Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites. Nature Communications, 10, 4176.

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Modulating Autophagy and Cell Signaling

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Dorsomorphin (AMPKi, S7306, 5 μM), SBI-0206965 (ULK1i, SML 1540, 10 μM), Caffeine (C0750, 2 mM), Ku55933 (ATM inhibitor, SML1109, 10 μM), 3-methyladenine (3-MA, 5142-23-4, 5 mM), cytochalasin D (C2618, 5 μg/ml), NCS (N9162, 0.5 mg/ml), HU (H8627, 2 mM), 7-hydroxy staurosporine (UCN-01, U6508, 100 nM), CPT (232120, 5 μM), pifithrin-α (p53 inhibitor, 506170, 10 μM), and chloroquine (CQ, 50-63-5, 50 μM) were purchased from Sigma, St. Louis, MO. Bafilomycin-A1 (Baf.A1, BML-CM110, 10 nM) was purchased from Enzo, NY, USA. Chk2 inhibitor II (220491, 10 μM) was purchased from Merck Millipore, Darmstadt, Germany. Akt inhibitor IV (124011, 5 µM) was purchased from Cell Signaling, Beverly, MA, USA. Cells were treated with all drugs for 24 h except HU, which added for 72 h to induce prolonged replication stress.

Chen T.Y., Huang B.M., Tang T.K., Chao Y.Y., Xiao X.Y., Lee P.R., Yang L.Y, & Wang C.Y. (2021). Genotoxic stress-activated DNA-PK-p53 cascade and autophagy cooperatively induce ciliogenesis to maintain the DNA damage response. Cell Death and Differentiation, 28(6), 1865-1879.

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Comprehensive Reagent Toolkit for Cell Death Studies

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The following drugs were used: Z-VAD-fmk (Promega, G7231), necrostatin-1 (Sigma, N9037), liproxstatin-1 (Sigma, SML1414), Fer-1 (Sigma, SML0583), N-acetyl-l-cysteine (Sigma, A7250), MitoTEMPO (Sigma, SML0737), deferoxamine mesylate salt (Merck, D9533), staurosporine (Sigma, S5921), erastin (Sigma, E7781), 1S,3R-RSL3 (Sigma, SML2234), iFSP1 (Sigma, SML2749), arachidonic acid (Sigma, 10931), FIN56 (Sigma, SML1740), sorafenib (Santa Cruz, sc-220125), inPISD inhibitor (or STK770988; Vitasmlab.biz), mitoquinol (Cayman Chemicals, 89950), CCCP (Sigma, C2759) and ULK1 inhibitor SBI-0206965 (Sigma, SML1540-5MG).

Zhu Y., Fujimaki M., Snape L., Lopez A., Fleming A, & Rubinsztein D.C. (2024). Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis. Nature Cell Biology, 26(4), 542-551.

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Culturing Primary Rat Cortical Neurons

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Primary rat cortical neurons were prepared from embryonic day 18 rats as described before [10 (link)] and cultured in cortex medium composed of serum-free neurobasal medium supplemented with B-27, penicillin/streptomycin/neomycin, l-glutamine (all Thermo Fisher Scientific, Waltham, MA, USA), and transferrin (AppliChem, Darmstadt, Germany) at 37 °C and 5% CO₂. On day in vitro (DIV) 1, cells were transduced with AAV.ULK1.DN and AAV.CTRL. Rapamycin (750 nM), staurosporine (30–300 nM), and SBI-0206965 (5 μM) or DMSO as control (all Sigma-Aldrich, St. Louis, MO, USA) were applied in selected conditions. For some experiments, medium samples were used for a bioluminescence-based toxicity assay (ToxiLight™, Lonza, Basel, Switzerland). Details of cell culture experiments can be found in the Supplementary Information (SI).

Vahsen B.F., Ribas V.T., Sundermeyer J., Boecker A., Dambeck V., Lenz C., Shomroni O., Caldi Gomes L., Tatenhorst L., Barski E., Roser A.E., Michel U., Urlaub H., Salinas G., Bähr M., Koch J.C, & Lingor P. (2020). Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing. Cell Death and Differentiation, 27(10), 2810-2827.

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Autophagy and Necroptosis Regulation Assays

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Capsaicin, chloroquine, rapamycin, and the ULK1 inhibitor (SBI-0206965) were obtained from Sigma-Aldrich (St. Louis, MO, United States). MG132 was purchased from TOCRIS Bioscience (Ellisville, MO, United States). Specific antibodies against ribophorin II and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Antibodies against poly (ADPribose) polymerase, caspase-3, phospho-elF2α, XBP1, ATG5, LC3B, phospho-MLKL (S358), and phospho-RIP3 (S227) were obtained from Cell Signaling Technology (Beverly, MA, United States). The antibody against p62 was purchased from Sigma-Aldrich. The antibody against phospho-IRE1α was obtained from Abcam (Cambridge, United Kingdom). The antibody against TRIB3 was obtained from Atlas Antibodies (Bromma, Sweden). The antibody against beclin 1 was purchased from Novus Biologicals (Centennial, CO, United States). The antibody against phospho-MLKL (T357/S358/S360) was purchased from ABclonal (Woburn, MA, United States).

Huang Y.C., Yuan T.M., Liu B.H., Liu K.L., Wung C.H, & Chuang S.M. (2021). Capsaicin Potentiates Anticancer Drug Efficacy Through Autophagy-Mediated Ribophorin II Downregulation and Necroptosis in Oral Squamous Cell Carcinoma Cells. Frontiers in Pharmacology, 12, 676813.

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