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Staurosporine

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Staurosporine is a small-molecule compound that inhibits a wide range of protein kinases. It is commonly used as a research tool in cell biology and biochemistry studies.

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Lab products found in correlation

Emetine dihydrochloride, by Merck Group (1 mentions) Valinomycin, by Merck Group (1 mentions) Doxazosin mesylate, by Merck Group (1 mentions) Infinite 200 pro series microplate reader, by Tecan (1 mentions) Dibutyryl camp, by Merck Group (1 mentions) Calcofluor white, by Merck Group (1 mentions) Caspase glo 3 7 assay kit, by Promega (1 mentions) Rapamycin, by AG Scientific (1 mentions)

4 protocols using staurosporine

1

Cytotoxicity Assay of Cardiomyocytes

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In order to assess the viability of the CMs to cytotoxic compounds, we performed a cell viability assay using CellTiter-Glo Luminescent Cell Vaibity Assay (Promega, Cat.#G7570). 96-well plates were coated with LN-521 and 15,000 CMs at day 34 were seeded into triplicate well. After 2 days of seeding, wells were washed and replenished with 90 mL of CMs maintenance medium. Cytotoxic drugs (valinomycin (Sigma, Cat.#94675), emetine dihydrochloride (Sigma, Cat.#E2375), doxazosin mesylate (Sigma, Cat.#D9815) and staurosporine (AG Scientific, Cat.#S-1016) were prepared in 10X maintenance medium containing 10% DMSO (Sigma, Cat.#D2650). Following that, 10 mL of each compound dilution was added to triplicate wells bringing the final volume to 100 mL in 1% DMSO for 24 hr. Cellular ATP concentrations were assessed using the assay kit as per manufacturer's instructions. Luminescence readings were taken on the Infinite 200 PRO series microplate reader (Tecan) at 1 s integration time.
Yap L., Wang J.W., Moreno-Moral A., Chong L.Y., Sun Y., Harmston N., Wang X., Chong S.Y., Vanezis K., Öhman M.K., Wei H., Bunte R., Gosh S., Cook S., Hovatta O., de Kleijn D.P.V., Petretto E, & Tryggvason K. (2019). In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors. Cell reports, 26(12).
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2

Culturing and Treating Candida albicans

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All C. albicans strains were archived in 25% glycerol and stored at −80°C. Overnight cultures were grown in YPD (1% yeast extract, 2% Bacto peptone, 2% dextrose) at 30°C. Two percent agar was added for solid media. Strains were constructed according to standard protocols. Strain construction is described in Text S1 in the supplemental material, and the strains used in this study are listed in Table S2. Staurosporine (AG Scientific, Inc., catalog no. S-1016) was formulated at 1 mg/ml in dimethyl sulfoxide (DMSO) and used at a final concentration of 0.5 μg/ml. Geldanamycin (Cedarlane catalog no. ant-gl-5) was formulated at 5 mM in DMSO and used at a final concentration of 10 μM. Hydroxyurea (BioShop Canada, Inc.) was formulated at 2 M in H2O, filter sterilized, and used at a final concentration of 200 mM. Dibutyryl cAMP (Sigma-Aldrich catalog no. D0627) was formulated at 100 mg/ml and used at a final concentration of 10 mg/ml. Fluorescent brightener 28, also known as calcofluor white (Sigma-Aldrich catalog no. F3543), was formulated at 10 mg/ml and used at a final concentration of 1 μg/ml.
Xie J.L., O’Meara T.R., Polvi E.J., Robbins N, & Cowen L.E. (2017). Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A. mSphere, 2(2), e00056-17.
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3

Caspase-3/7 Activation in L. monocytogenes-Infected Macrophages

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iIFNAR−/− macrophages were seeded into white 96-well plates and infected with L. monocytogenes at an MOI of 10. Staurosporine (AG Scientific) (5 µM) was added to uninfected macrophages at 30 min postinfection to induce caspase-3/7 activation. At 6 h postinfection, caspase-3/7 activation was measured using a caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s instructions.
Chen G.Y., McDougal C.E., D’Antonio M.A., Portman J.L, & Sauer J.D. (2017). A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival. mBio, 8(2), e00119-17.
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4

Yeast Growth Assay with Small Molecules

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Latrunculin B, Rapamycin, Staurosporine, and Tacrolimus were purchased from AG Scientific. All other drugs were purchased from Sigma. All drugs were dissolved in DMSO (except bromopyruvate and sodium azide, which were dissolved in water) and kept at −20°C. All experiments were conducted with S. cerevisiae strains BY4741 or BY4743 or heterozygous/heterozygous deletion strain collections derived from BY4743. Yeast cells were grown in yeast extract peptone dextrose (YPD) (1% yeast extract, 2% bacto-peptone, and 2% glucose) overnight (OD600~5) and diluted to an OD600 of 0.01 in YPD with the desired drug concentrations controlled for solvent concentrations (plus 0.2 M NaCl in Tac+SFK experiment). Cells were grown at 30°C for 20–24 hr in 96-well plates in Tecan F200 or Genios microplate readers, with OD595 readings of cell density recorded every 15 min. We used the area under growth curve (AUC) of each condition as a metric of cell growth, after discarding the first ten measurement points. The growth measurement data obtained during this study are given as Table S2.
Cokol M., Weinstein Z.B., Yilancioglu K., Tasan M., Doak A., Cansever D., Mutlu B., Li S., Rodriguez-Esteban R., Akhmedov M., Guvenek A., Cokol M., Cetiner S., Giaever G., Iossifov I., Nislow C., Shoichet B, & Roth F.P. (2014). Large-Scale Identification and Analysis of Suppressive Drug Interactions. Chemistry & biology, 21(4), 541-551.
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