Mouse ES cells (JM8.N4, RRID: CVCL_J962) were used for all experiments and obtained as previously described (15 (link)). ES cells were cultured on 0.1% gelatin-coated plates in ESC DMEM (Corning 10101CV, contains 4.5 g/l glucose, 110 mg/l sodium pyruvate, glutagro, and 15 mg/l phenol red) with 15% FBS (HyClone), 0.1 mM MEM non-essential amino acids (Gibco), 2 mM l -glutamine (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 1× Penicillin Streptomycin solution (Corning), and 1000 units/ml of ESGRO LIF (Chem-icon). ESCs were fed daily, cultured at 37°C in a 5% CO2 incubator, and passaged every 2 days by trypsinization. For Leptomycin B treatment, cells were treated with 10 ng/ml Leptomycin B (Sigma) at 37°C in a 5% CO2 incubator for 1 hour. For all imaging experiments, cells were imaged in DMEM without phenol red (Gibco 31053028, contains 4.5 g/l glucose) with 110 mg/l sodium pyruvate (Gibco) and all other components previously listed.
Leptomycin b
Manufactured by Merck Group
Sourced in United States
Leptomycin B is a laboratory reagent produced by Merck Group. It is a natural product isolated from Streptomyces bacteria. Leptomycin B functions as a nuclear export inhibitor, which can be used in research applications to study cellular processes involving nuclear export.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (11 mentions)
Cycloheximide (chx), by Merck Group (7 mentions)
Actinomycin d, by Merck Group (5 mentions)
Nocodazole, by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Trichostatin a, by Merck Group (3 mentions)
Ivermectin, by Merck Group (3 mentions)
Ly294002, by Merck Group (3 mentions)
Importazole, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Reversine, by Merck Group (2 mentions)
Flavopiridol, by Bio-Techne (2 mentions)
Latrunculin b, by Merck Group (2 mentions)
Exportin, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Q vd oph qvd, by Merck Group (2 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Verikine human ifn β elisa kit, by PBL Assay Science (2 mentions)
Il 1β elisa kit, by RayBiotech (2 mentions)
86 protocols using leptomycin b
1
Mouse Embryonic Stem Cell Culture
2
Selective Protein Knockdown in Cell Lines
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NIH3T3, HEK293T, PC6-3, and A549 cells were cultured and transfected as described (Strack et al., 2004 (link); Jin et al., 2010 (link)). siRNA-mediated knockdown of endogenous B56ε in NIH3T3 or knockdown of endogenous Fam13a in A549 cells was performed using Lipofectamine 2000 transfection reagent (Life Technologies, Gaithersburg, MD). The sequences for the sense and antisense strands of siRNAs are as follows: siGFP, 5′-gcaagcugacccugaaguucuu-3′; 5′-gaacuucagggucagcuugcuu-3′. siB56ε, 5′-ccuagugacagcaaugaauuu-3′; 5′ -auucauugcugucacuagguu-3′. siFam13a, 5′-ggagaacucuuagaaagaauu-3′; 5′-uucuuucuaagaguucuccuu-3′. Knockdown of B56s in PC6-3 cells was carried out as described (Strack et al., 2004 (link); Jin et al., 2011 (link)). For PP2A and Akt inhibition studies, NIH3T3 cells were treated with okadaic acid (50 nM, #O7885; Sigma-Aldrich, St. Louis, MO) and/or wortmannin (2 μM, #W1628; Sigma-Aldrich) or LY294002 (#L9908; Sigma-Aldrich) in DMEM supplemented with 0.5% bovine calf serum 1 d after transfection and harvested 15 h after drug administration. For leptomycin B treatment, NIH3T3 cells were exposed to 5 ng/ml leptomycin B (#L2913; Sigma-Aldrich) and/or 50 nM okadaic acid for 15 h before fixation.
3
Molecular Pathways Modulation in Cells
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For the treatment with MG132 proteasome inhibitor: U2OS cells were treated either with DMSO or 10 μM MG132 (Sigma-Aldrich) for 5 hours. Cells were harvested and total cell extracts were prepared for subsequent analysis as described [8 ].
Treatment with Bisindolylmalemide or TPA was performed as follows: 24 hours after transfection, HeLa cells were treated either with DMSO or Bisindolylmalemide (Calbiochem) at 10 μM final concentration for 16 hours or TPA (Applichem) at 10 μM final concentration for 5 minutes. Cells were harvested and total extracts prepared for subsequent analysis as described.
Treatment with Leptomycin B: 24 hours after transfection, U2OS cells were treated either with Methanol or with Leptomycin B (Sigma-Aldrich) at 10 μM final concentration for 4 hours. Cells were harvested and total extracts prepared for subsequent analysis as described.
Treatment with Bisindolylmalemide or TPA was performed as follows: 24 hours after transfection, HeLa cells were treated either with DMSO or Bisindolylmalemide (Calbiochem) at 10 μM final concentration for 16 hours or TPA (Applichem) at 10 μM final concentration for 5 minutes. Cells were harvested and total extracts prepared for subsequent analysis as described.
Treatment with Leptomycin B: 24 hours after transfection, U2OS cells were treated either with Methanol or with Leptomycin B (Sigma-Aldrich) at 10 μM final concentration for 4 hours. Cells were harvested and total extracts prepared for subsequent analysis as described.
4
Profiling Cellular Stress Responses
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HeLa (ATCC, CCL-2), Hap1 (Horizon Discovery, Cambridge, UK), and Hap1-derived Nup-mEGFP cell lines (this paper) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Waltham, MA), supplemented with l-glutamine and sodium pyruvate (from here-on referred to as DMEM) and 10% (vol/vol) fetal bovine serum (FBS, Sigma, St. Louis, MO) in humidified incubators at 37C and in a 5% pCO2 atmosphere, using standard sterile techniques. HeLa cells were recently acquired from ATCC and Hap1 cell lines were acquired from Horizon Discovery. Cells were negative for mycoplasma.
For starvation experiments, cells were imaged, then washed 3x with PBS and placed in 1x Hank’s Balanced Salt Solution with calcium and magnesium (HBSS, Gibco) for 24 hr and imaged. For leptomycin B experiments, cells were either treated with 25 nM leptomycin B (Sigma) or vector (methanol) for 15 hr, at which point both were imaged.
For starvation experiments, cells were imaged, then washed 3x with PBS and placed in 1x Hank’s Balanced Salt Solution with calcium and magnesium (HBSS, Gibco) for 24 hr and imaged. For leptomycin B experiments, cells were either treated with 25 nM leptomycin B (Sigma) or vector (methanol) for 15 hr, at which point both were imaged.
5
Subcellular Fractionation and Western Blot Analysis
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U-251, HCT-116 or HeLa cells were plated in 100mm dishes and subcellular fractionations were performed as previously described31 (link). Briefly, cells were centrifuged for 5 minutes at 1500 rpm, then lysed in buffer A (10mM HEPES, pH 7.5, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1mM DTT and complete protease inhibitors (Roche)), and then incubated for 15 min on ice, 10% NP-40 was added followed by vigorous vortexing, nuclei were spun down by centrifugation for 5 minutes at 3000 rpm. The pellet was re-suspended in buffer C (20mM HEPES, pH 7.5, 420 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 20% glycerol, 1 mM DTT and complete protease inhibitors (Roche)) and left for 30 minutes on ice followed by 13000 rpm for 30 minutes. Subcellular fractions were dissolved in/supplemented with Laemmli sample-loading buffer and subjected to 4–12% SDS–PAGE followed by Western Blot analysis with antibodies against FLAG M2 (Sigma), MTHFD2 (Abcam), Lamin A/C, COXIV and α-Tubulin (Pierce). For experiments involving transient transfection and treatment with 10 ng/mL Leptomycin B (Sigma), cells were seeded 24 hours before transfection and allowed to recover 24 hours before Leptomycin B treatment for 12 hours.
6
Inhibiting Ribosome Biogenesis and Proteasome
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To inhibit ribosome biogenesis, cells were treated with 20 µg/µL Leptomycin B (Merck, 431050) for 2 days, unless otherwise specified. To inhibit proteasome degradation, cells were treated with 10 µM MG132 (Sigma, M8699) for at least 2 h. To induce oxidative stress, cells were incubated with 1 mM or 0.1 mM H2O2 (AlfaAesar L13235) for 10 min.
7
Ribosome Biogenesis and Oxidative Stress
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To inhibit ribosome biogenesis, cells were treated with 20 µg/µL Leptomycin B (Merck, 431050) for 2 days, unless otherwise specified. To inhibit proteasome degradation, cells were treated with 10 µM MG132 (Sigma, M8699) for at least 2 hr. To induce oxidative stress, cells were incubated with 1mM or 0.1mM H2O2 (AlfaAesar L13235) for 10 min.
8
Mitotic Arrest and Exit Protocols
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To arrest cells in prometaphase, cells were incubated for 2 – 4 h in 200 ng ml−1 nocodazole (Sigma). Acute spindle depolymerization was induced with 3 μg ml−1 nocodazole. To arrest cells in metaphase, cells were incubated for 30 min in 10 μM MG132 (Sigma Aldrich). Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM. Exportin-1/CRM1 was inhibited by 2 h incubation in leptomycin B (Sigma Aldrich), final concentration 1 μg ml−1. Actin was depolymerized by 2 h incubation in 1 μM latrunculin B (Sigma Aldrich). For viability measurements TO-PRO-3 Iodide (Molecular Probes) was used at a final concentration of 1 μM. Rapamycin (Calbiochem 5S3210) was used at a final concentration of 500 nM. Trichostatin A (Sigma T8552) was added 2 h before imaging at a final concentration of 0.5 μM.
9
Nitrogen Starvation and Response Imaging
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Seedlings were grown for 5 d on the 3 mM KNO 3 liquid medium (see above), nitrogen-starved for 2 d by exchanging the culture medium with a nitrogen-free medium, in which KNO 3 was replaced by KCl. Plants were mounted in 40 μL of a top agar (0.1%, w/v) solution. During confocal imaging, either 5 μL of 0.5 M KNO 3 , 5 μL of 0.5 M NH4Cl, were added to the mounting medium; the same plantlets were imaged during a time course. For the nuclear export inhibition experiment, a 3-h preincubation with Leptomycin B (Sigma, at 100 nM final concentration) was performed in N-free medium.
Laser scanning confocal imaging was performed using the Zeiss 710 or the Leica SP8 microscope equipped with an argon laser (488 nm for GFP excitation and 561 nm for mCherry). Emission was collected at 510-545 nm (GFP) and at 600-630 nm (mCherry). A sequential scanning mode was used when mCherry was combined with GFP to minimize the crosstalk between the two partially overlapping emission spectra. The images were coded green for GFP and magenta for mCherry giving white colocalization in merged images. Images were processed in ImageJ and Photoshop Element (Adobe Systems). Each image shown represents a single focal plane.
Laser scanning confocal imaging was performed using the Zeiss 710 or the Leica SP8 microscope equipped with an argon laser (488 nm for GFP excitation and 561 nm for mCherry). Emission was collected at 510-545 nm (GFP) and at 600-630 nm (mCherry). A sequential scanning mode was used when mCherry was combined with GFP to minimize the crosstalk between the two partially overlapping emission spectra. The images were coded green for GFP and magenta for mCherry giving white colocalization in merged images. Images were processed in ImageJ and Photoshop Element (Adobe Systems). Each image shown represents a single focal plane.
10
Inhibition of Cell Signaling Pathways
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LiCl (GSK3β inhibitor), MG132 (Proteasome inhibitor), PD98059 (ERK1/2 inhibitor), SB230580 (p38 inhibitor), leptomycin B (LMB, Nuclear export inhibitor), zinc protoporphyrin IX (ZnPP, HO-1 inhibitor), 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Fluorouracil (5-FU) and oxaliplatin were purchased in Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, p-GSK3β, total-GSK3β, p-p38, total-p38, HO-1, Nrf2, cleaved PARP, TBP and β-actin were purchased in Cell Signaling (Bervely, MA, USA).
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