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Latrunculin b

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Latrunculin B is a natural marine macrolide compound that functions as an actin-disrupting agent. It binds to actin monomers, preventing their polymerization and leading to the depolymerization of actin filaments. This disruption of the actin cytoskeleton can have various effects on cellular processes.

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Lab products found in correlation

Zstk474, by Cell Signaling Technology (4 mentions) Y 27632, by Enzo Life Sciences (3 mentions) Gefitinib, by Cayman Chemical (3 mentions) Pf562271, by Adipogen (3 mentions) Ck 666, by Merck Group (2 mentions) Rapamycin, by Cayman Chemical (2 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Ionomycin, by Thermo Fisher Scientific (2 mentions) Yohimbine, by Merck Group (2 mentions) Jasplakinolide, by Cayman Chemical (2 mentions) Dasatinib, by Cayman Chemical (2 mentions) Anisomycin, by Merck Group (2 mentions) Bapta am, by Selleck Chemicals (2 mentions) Ly2584702, by Selleck Chemicals (2 mentions) Gdc 0994, by Apexbio (2 mentions) Uk14304, by Merck Group (2 mentions) α factor, by GenWay Biotech (1 mentions) Latrunculin a, by Enzo Life Sciences (1 mentions) Pp242, by Merck Group (1 mentions) Jasplakinolide, by Enzo Life Sciences (1 mentions)

11 protocols using latrunculin b

1

Modulation of Cytoskeletal Dynamics in Cell Irradiation

Cited 3 times
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Myosin and actin polymerization inhibitors were added to the cultures before IR treatments. In Drosophila cells, treatment conditions were: 2 mM or 10 mM BDM (Sigma, Ref. 63 (link)) for 5 min; 200 or 400 μM MyoVin-1 (Calbiochem, Ref. 64 (link)) for 2 h; 100 μM or 400 μM CK666 (Sigma, Ref. 65 (link)) for 60 min; 4 μM or 8 μM Latrunculin B (Enzo Life Sciences) for 20 min. Stocks of MyoVin-1 (100 mM), CK666 (100 mg/ml) and Latrunculin B (10 mM) were prepared in DMSO and those of BDM were diluted in water. For mouse cells, treatment conditions were: 7.5 mM BDM for 20 min; 200 μM MyoVin-1 for 60 min; 400 μM CK666 for 60 min; 8 μM Latrunculin B for 20 min. All stocks were prepared in DMSO. For controls, DMSO was added to the media to yield final concentrations equivalent to samples treated with chemicals. For the ‘release’ time points of Extended Data Figs 1f–g and 6h, cells were washed 3× after incubation with each chemical, and incubated at 27 °C for 1 h before IR. Cells were fixed 60 min after IR. Notably, the doses of LatB treatments used affect nuclear actin polymerization in addition to cytoplasmic actin66 (link).
Caridi C.P., D’Agostino C., Ryu T., Zapotoczny G., Delabaere L., Li X., Khodaverdian V.Y., Amaral N., Lin E., Rau A, & Chiolo I. (2018). Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature, 559(7712), 54-60.
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2

Cell cycle arrest and imaging

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Cells were grown to mid log phase in CSM + dextrose medium at 30°C, arrested with 2 μM α-factor (Genway Biotech, San Diego, CA) for 3 h, washed, and released into fresh CSM + dextrose. For the experiment of Figure 2, α-factor was added back 1 h after release so that cells would rearrest after a single cycle. Samples were taken, and budding percentage was scored at 15-min intervals.
For the experiment of Figure 8, cells were mounted onto a slab with 100 μM latrunculin B (Enzo Life Sciences, Farmingdale, NY) after a 10-min recovery period in fresh medium after release from arrest. Images were acquired as stated but at 2-min instead of 1.5-min intervals.
Kang H., Tsygankov D, & Lew D.J. (2016). Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1. Molecular Biology of the Cell, 27(11), 1764-1775.
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3

Modulation of Cytoskeletal Dynamics in Cell Irradiation

Cited 3 times
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Myosin and actin polymerization inhibitors were added to the cultures before IR treatments. In Drosophila cells, treatment conditions were: 2 mM or 10 mM BDM (Sigma, Ref. 63 (link)) for 5 min; 200 or 400 μM MyoVin-1 (Calbiochem, Ref. 64 (link)) for 2 h; 100 μM or 400 μM CK666 (Sigma, Ref. 65 (link)) for 60 min; 4 μM or 8 μM Latrunculin B (Enzo Life Sciences) for 20 min. Stocks of MyoVin-1 (100 mM), CK666 (100 mg/ml) and Latrunculin B (10 mM) were prepared in DMSO and those of BDM were diluted in water. For mouse cells, treatment conditions were: 7.5 mM BDM for 20 min; 200 μM MyoVin-1 for 60 min; 400 μM CK666 for 60 min; 8 μM Latrunculin B for 20 min. All stocks were prepared in DMSO. For controls, DMSO was added to the media to yield final concentrations equivalent to samples treated with chemicals. For the ‘release’ time points of Extended Data Figs 1f–g and 6h, cells were washed 3× after incubation with each chemical, and incubated at 27 °C for 1 h before IR. Cells were fixed 60 min after IR. Notably, the doses of LatB treatments used affect nuclear actin polymerization in addition to cytoplasmic actin66 (link).
Caridi C.P., D’Agostino C., Ryu T., Zapotoczny G., Delabaere L., Li X., Khodaverdian V.Y., Amaral N., Lin E., Rau A, & Chiolo I. (2018). Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature, 559(7712), 54-60.
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4

Preparation and Handling of Common Pharmacological Agents

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Stocks of 10 mM PP242 (Sigma, #P0037), 25 mM Latrunculin A (Enzo, #BML-T119–0100), 10 mM Ulixertinib (MedChemExpress, #HY-15816), 50 mM LY294002 (Invitrogen, #PHZ1144), 10 mM ZSTK474 (Cell Signaling, #13213), 10 mM Y27632 (Enzo, #ALX-270–333-M001), 25 mM Latrunculin B (Enzo, #BML-T110–0001), 1 mM Jasplakinolide (Enzo, #ALX-350–275-C050) and 10 mM Rapamycin (Cayman, #13346) were prepared by dissolving the chemicals in DMSO. The stocks were diluted to the indicated final concentrations in culture medium or live cell imaging medium. The EGF stock solution was prepared by dissolving EGF (Sigma, #E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. Insulin (Sigma #I-1882) was resuspended at 10 mg/ml in sterile ddH2O containing 1% glacial acetic acid. Hydrocortisone (Sigma #H-0888) was resuspended at 1 mg/ml in 200 proof ethanol. Cholera toxin (Sigma #C-8052) was resuspended at 1 mg/ml in sterile ddH2O and stored at 4°C. All drug stocks except Cholera toxin were stored at −20°C.
Zhan H., Bhattacharya S., Cai H., Iglesias P.A., Huang C.H, & Devreotes P.N. (2020). An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells. Developmental cell, 54(5), 608-623.e5.
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5

Chemical Compound Preparation Protocol

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Stocks of 200 μM phorbol-12,13-dibutyrate (PDBu, EMD Millipore, #524390), 1 mg/mL anisomycin (Sigma-Aldrich, A9789), 10 mM UK14304 (Sigma-Aldrich, U104), 10 mM yohimbine (Sigma-Aldrich, Y3125), 1 mM gefitinib (Cayman, #13166), 1 mM ionomycin (Peprotech, #5608212), 2 mM jasplakinolide (Cayman, #11705), 25 mM latrunculin B (Enzo Life Sciences, BML-T110–0001), 10 mM PF562271 (AdipoGen, SYN-1064), 10 mM ZSTK474 (Cell Signaling, #13213), 10 mM dasatinib (Cayman, #11498), 10 mM GDC-0994 (APExBIO Technology, B5817), 1 mM LY2584702 (Selleck, S7698), 10 mM BAPTA-AM (Selleck, S7534) were prepared by dissolving the chemicals in DMSO; 10 mM Y27632 (Enzo Life Sciences, #ALX-270–333) in water. Stocks were diluted to the indicated final concentrations in the culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma-Aldrich, E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. All drug stocks were stored at −20°C. 2-Deoxyglucose (2-DG, MilliporeSigma, #D8375) was dissolved in culture medium to 100 mM and used immediately.
Yang J.M., Chi W.Y., Liang J., Takayanagi S., Iglesias P.A, & Huang C.H. (2021). Deciphering cell signaling networks with massively multiplexed biosensor barcoding. Cell, 184(25), 6193-6206.e14.
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6

Caspase-1 Activation in Monocytes

Cited 1 time
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To measure spontaneous or LPS-induced activation and secretion of caspase-1 in supernatants of monocytes, day 4 BM-derived cells (2 × 106) were seeded in a 12-well plate in 1 ml of medium (DMEM containing 10% FCS). Cells were then left untreated for 4 h, washed with serum-free medium (DMEM), and incubated for 44 h or stimulated with 1 µg/ml LPS for 4 h, washed with DMEM, and incubated with or without 10 µM nigericin (Enzo Life Sciences) in DMEM for 44 h. Additionally, wild-type and Wdr1rd/rd cells were pretreated with 1 µM latrunculin-b (Enzo Life Sciences) or 1 µM colchicine (Sigma-Aldrich) for 30 min before LPS priming for 4 h, washed with DMEM, and incubated for 44 h with or without latrunculin-b or colchicine. Culture supernatants were then collected and enriched by methanol chloroform precipitation (Masters et al., 2010 (link)). Precipitates were boiled for 5 min with SDS sample buffer, resolved by SDS/PAGE, and transferred to polyvinylidene fluoride membranes. After blocking in PBS with 0.1% Tween 20 and 5% skim milk for 1 h, membranes were then probed overnight at 4°C with anti–caspase-1 (p20; AdipoGen).
Kim M.L., Chae J.J., Park Y.H., De Nardo D., Stirzaker R.A., Ko H.J., Tye H., Cengia L., DiRago L., Metcalf D., Roberts A.W., Kastner D.L., Lew A.M., Lyras D., Kile B.T., Croker B.A, & Masters S.L. (2015). Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1β. The Journal of Experimental Medicine, 212(6), 927-938.
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7

Cell Culture and Manipulation Techniques

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COS1 and HEK 293T cells were maintained at 37°C and a 5% CO2 atmosphere in DMEM supplemented with 10% fetal bovine serum (FBS) plus 1% l-glutamine plus 100 U/ml of both penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO). When indicated, COS1 cells were incubated with 2 μM cytochalasin D (15 min; Sigma-Aldrich), 10 μM latrunculin B (20 min; Enzo Life Sciences, Farmingdale, NY), or 50 μM blebbistatin (2 h; Sigma-Aldrich). Jurkat cells were maintained as described in RPMI-1640 supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. When necessary, Jurkat cells were stimulated for the indicated periods of time with antibodies to human CD3 (10 μg/ml; Castro-Castro et al., 2011 (link); Barreira et al., 2014 ). The stable CORO1A-knockdown COS1 (KD1A.1 cell clone) and Jurkat (J.KD1A cell pool) cells were generated, characterized, and cultured as described elsewhere (Castro-Castro et al., 2011 (link); Ojeda et al., 2014 (link)).
Ojeda V., Robles-Valero J., Barreira M, & Bustelo X.R. (2015). The disease-linked Glu-26-Lys mutant version of Coronin 1A exhibits pleiotropic and pathway-specific signaling defects. Molecular Biology of the Cell, 26(16), 2895-2912.
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8

Preparation of Pharmacological Reagents for Cell-Based Assays

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Stocks of 50 mM LY294002 (Cayman, #70920), 5 mM Latrunculin A (AdipoGen, #AGCN20027C100), 25 mM Latrunculin B (Enzo, #BML-T110-0001), 10 mM MEK inhibitor PD325901 (Calbiochem, #444966), 10 mM FAK inhibitor PF-573228 (Cayman, #14924), 10 mM FAK inhibitor PF-562271 (AdipoGen, SYN-1064), 10 mM ZSTK474 (Cell Signaling, #13213), 1 mM Gefitinib (Cayman, #13166), and 10 mM Rapamycin (Cayman, #13346) were prepared by dissolving the chemicals in DMSO. The stocks were diluted to the indicated final concentrations in culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma, #E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. All drug stocks were stored at −20 °C.
Yang J.M., Bhattacharya S., West-Foyle H., Hung C.F., Wu T.C., Iglesias P.A, & Huang C.H. (2018). Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation. Nature Communications, 9, 4673.
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9

Chemical Compound Preparation Protocol

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Stocks of 200 μM phorbol-12,13-dibutyrate (PDBu, EMD Millipore, #524390), 1 mg/mL anisomycin (Sigma-Aldrich, A9789), 10 mM UK14304 (Sigma-Aldrich, U104), 10 mM yohimbine (Sigma-Aldrich, Y3125), 1 mM gefitinib (Cayman, #13166), 1 mM ionomycin (Peprotech, #5608212), 2 mM jasplakinolide (Cayman, #11705), 25 mM latrunculin B (Enzo Life Sciences, BML-T110–0001), 10 mM PF562271 (AdipoGen, SYN-1064), 10 mM ZSTK474 (Cell Signaling, #13213), 10 mM dasatinib (Cayman, #11498), 10 mM GDC-0994 (APExBIO Technology, B5817), 1 mM LY2584702 (Selleck, S7698), 10 mM BAPTA-AM (Selleck, S7534) were prepared by dissolving the chemicals in DMSO; 10 mM Y27632 (Enzo Life Sciences, #ALX-270–333) in water. Stocks were diluted to the indicated final concentrations in the culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma-Aldrich, E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. All drug stocks were stored at −20°C. 2-Deoxyglucose (2-DG, MilliporeSigma, #D8375) was dissolved in culture medium to 100 mM and used immediately.
Yang J.M., Chi W.Y., Liang J., Takayanagi S., Iglesias P.A, & Huang C.H. (2021). Deciphering cell signaling networks with massively multiplexed biosensor barcoding. Cell, 184(25), 6193-6206.e14.
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10

Comprehensive Antibody Validation for Cellular Analysis

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Primary antibodies against following proteins were used: CAP1 (Abcam ab133655), CD36 (Santa Cruz H-300 sc-9154), Syk (Santa Cruz 4D10 sc-1240), GAPDH (Calbiochem 6C5-CB1001), β-actin (Abcam ab20272), β3-integrin (Santa Cruz HC93 sc-14009), cofilin (Cell Signaling Technology D3F9 #5175), profilin-1 (Cell Signaling Technology #3237), and vinculin (Sigma SAB4200080). Secondary antibodies Alexa Fluor 568- or 488-conjugated anti-mouse and anti-rabbit immunoglobulins (Molecular Probes, Invitrogen Life Technologies Ltd.) were used for immunofluorescence. Peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulins (Sigma-Aldrich Co. Ltd.) or IRDye 680 or IRDye 800 anti-mouse and anti-rabbit immunoglobulins (LI-COR Biosciences, Lincoln, USA) were used for Western blot.
Human fibrinogen was from Enzyme Research (Swansea, UK), collagen (Kollagenreagens Horm) was from Takeda (Osaka, Japan), recombinant human resistin (450-19) was from PeproTech (London, UK), latrunculin B and GSNO were from Enzo Life Sciences (Exeter, UK). PGI2 was from Cayman Chemical (Michigan, USA). Glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 was from Abcam. Other reagents were from Sigma-Aldrich unless otherwise indicated.
Joshi P., Riley D.R., Khalil J.S., Xiong H., Ji W, & Rivero F. (2018). The membrane-associated fraction of cyclase associate protein 1 translocates to the cytosol upon platelet stimulation. Scientific Reports, 8, 10804.
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