erastin (Bio-techne, 5449); SAS (MedchemExpress, HY-14655); RSL3 (Cayman, 19288); FIN56 (Cayman, 25180); FINO2 (MedchemExpress, HY-129457); PANKi (Cayman, 31002); BSO (Sigma, B2515); DEM (Sigma, D97703); etomoxir sodium salt (Selleckchem, S8244); lovastatin (Selleckchem, S2061); TOFA (Selleckchem, S6690); dorsomorphin (Selleckchem, S7306); alisertib (Selleckchem, S1133); verdinexor (Cayman, 26171); leptomycin B (Cayman, 10004976); tipifarnib (MedchemExpress, HY-10502); methotrexate (Selleckchem, S1210); pitstop2 (Sigma, SML1169); EML425 (Selleckchem, S2977); C646 (Selleckchem, S7152); brequinar (Selleckchem, S3565); elamipretide (Selleckchem, S9803)
Leptomycin b
Manufactured by Cayman Chemical
Sourced in United States
Leptomycin B is a small molecule compound produced by the bacterium Streptomyces. It is a potent inhibitor of the nuclear export receptor protein Exportin 1 (XPO1/CRM1). Leptomycin B is commonly used as a research tool to study nucleocytoplasmic transport and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (5 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti nrf2 d1z9c, by Cell Signaling Technology (2 mentions)
Mg132, by Santa Cruz Biotechnology (2 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (2 mentions)
Kt5720, by Merck Group (2 mentions)
Doxycycline hyclate, by Merck Group (2 mentions)
Pitstop 2, by Merck Group (1 mentions)
Erastin, by Bio-Techne (1 mentions)
Lovastatin, by Selleck Chemicals (1 mentions)
Verdinexor, by Cayman Chemical (1 mentions)
Tipifarnib, by MedChemExpress (1 mentions)
Methotrexate, by Selleck Chemicals (1 mentions)
Brequinar, by Selleck Chemicals (1 mentions)
Alisertib, by Selleck Chemicals (1 mentions)
Fin56, by Cayman Chemical (1 mentions)
Dorsomorphin, by Selleck Chemicals (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Mln4924, by Selleck Chemicals (1 mentions)
12 protocols using leptomycin b
1
Ferroptosis Inducers and Inhibitors Screening
2
Antibody Sources and Chemical Reagents
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Antibodies against Beta-ACTIN (C-4), BACH1 (F-9) and LAMIN B2 (C-20) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-NRF2 (D1Z9C) was obtained from Cell Signalling Technology (Danvers, MA, USA) and anti-HMOX1 was purchased from Biovision (San Francisco, CA, USA). Antibody against ALPHA-TUBULIN was obtained from Sigma-Aldrich (St. Louis, MO, USA). HRP-conjugated secondary antibodies were obtained from Life Technologies (Carlsbad, California, USA). Dimethyl sulfoxide (DMSO) was from Sigma-Aldrich. R,S-sulforaphane (SFN) was purchased from LKT Laboratories (St. Paul, MN, USA). (±)-TBE-31 was synthesized as described [41 ,42 (link)]. CDDO and CDDO-derivatives were obtained from Cayman Chemicals (Ann Arbor, MI, USA). MG132 was obtained from Santa Cruz Biotechnology, Leptomycin B from Cayman Chemicals, MLN4924 and Selinexor (KPT-330) from Selleckchem (Houston, TX, USA) and Actinomycin D and Cycloheximide from Sigma.
3
Rodent Primary Cortical Neuron Preparation
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Rodent primary cortical neurons were prepared as described in18 (link). Briefly, neurons dissected from embryonic day 20-21 Long Evans rat pups were cultured at a density of 0.6 × 106 cells ml-1 in 96 well plates coated with laminin (Corning) and D-polylysine (Millipore). Euthanasia for these experiments is entirely consistent with the recommendations of the Guidelines on Euthanasia of the American Veterinary Medical Association. Primary neurons were transfected with plasmids and/or siRNA (obtained from Dharmacon) 4 days after plating using Lipofectamine 2000 (Invitrogen)21 (link). SINE compounds KPT-335 and KPT-350 (Karyopharm Therapeutics), Leptomycin B (Cayman Chemicals) or vehicle (DMSO, Sigma) were added 24 h after transfection, immediately following the first imaging run. In experiments measuring subcellular localization of fluorescent proteins, the nucleus was identified using cell-permeable nuclear markers (Hoechst 33342, Sigma).
HEK293 cells were passaged every 3-4 days (~90% confluence) using trypsin. For transfection, HEK293 cells were passaged 1:10 and transfected 24 h later using Lipofectamine2000 (Invitrogen), according to the manufacturer’s protocol, and cell-permeable nuclear marker DRAQ5 (Thermo Fisher) added post-transfection.
HEK293 cells were passaged every 3-4 days (~90% confluence) using trypsin. For transfection, HEK293 cells were passaged 1:10 and transfected 24 h later using Lipofectamine2000 (Invitrogen), according to the manufacturer’s protocol, and cell-permeable nuclear marker DRAQ5 (Thermo Fisher) added post-transfection.
4
Evaluation of Proteasome Inhibitors in Myeloma
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Antibodies against IκBα (C-21), IκBβ (C-20) were obtained from Santa Cruz Biotechnology and antibody against tubulin (CP06) purchased from Calbiochem, recombinant human TNFα (654205, EMD Millipore), cycloheximide (C7698, Sigma-Aldrich), and leptomycin B (87081-35-4, Cayman Chemicals). Bortezomib (S1013), ixazomib citrate (S2181), carfilzomib (S2853), melphalan (S8266), and bendamustine (S1212) were purchased from Selleckchem. Dexamethasone (D4902), doxorubicin (D1515), and lenalidomide (CDS022536) were purchased from Sigma-Aldrich. Selinexor (KPT-330) was provided by Dr. Trinayan Kashyap (Karyopharm). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (M6494) was purchased from Thermo-Fisher Scientific.
5
Leptomycin B and KPT 350 in Fly Lifespan
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Leptomycin B (Cayman Chemical) was dissolved in 100% ethanol. KPT 350 was a gift from Karyopharm Therapeutics (Newton, MA, USA) and dissolved in DMSO. On the day of eclosion (day 0), flies were transferred to food containing 500 nM of drug or the appropriate vehicle. Flies were flipped to fresh vials of drug or vehicle‐treated food twice a week. Ten days post‐eclosion, flies were collected and fixed in formalin for TUNEL staining or frozen at −80°C for Western blotting.
6
Ago2 Nuclear Localization Assay
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2 × 105 HeLa cells or stable monoclonal HeLa FH-NLS-Ago2 were plated on cover slips in 24-well plates one day before assay. Two hours before addition of NIH 3T3 cells, medium was changed to DMEM + 1% FBS + P/S + 75 μg/ml cycloheximide (Sigma–Aldrich). 4 × 104 cells NIH 3T3 were added to each well and co-cultured with HeLa for 2–4 h until attachment. Cells were then treated with 1 ml PBS + 50% (w/v) polyethylene glycol 1500 (Sigma–Aldrich) for 2 min at room temperature followed by five washing steps with PBS. Then, DMEM + 1% FBS + P/S + 75 μg/ml cycloheximide was added again to cells for 4 h. Assay was stopped by adding PBS + 4% paraformaldehyde. For detection of endogenous Ago2 or FH-NLS-Ago2, cells were used for immunofluorescence staining as described above.
Leptomycin B was purchased from Cayman Chemicals as 0.1 μg/μl solution in ethanol. Leptomycin B was added to cells to 10 ng/ml final concentration in standard cultivation medium. Ethanol was added to control cells without Leptomycin B. Treatments were done for 4 h and stopped by addition of PBS + 4% paraformaldehyde and subsequent immunofluorescence staining.
Leptomycin B was purchased from Cayman Chemicals as 0.1 μg/μl solution in ethanol. Leptomycin B was added to cells to 10 ng/ml final concentration in standard cultivation medium. Ethanol was added to control cells without Leptomycin B. Treatments were done for 4 h and stopped by addition of PBS + 4% paraformaldehyde and subsequent immunofluorescence staining.
7
Bicarbonate Stimulation of Melanoma Cells
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Drugs were prepared according to manufacturers’ instructions and added directly to cell culture medium. Doxycycline hyclate (Sigma, #D9891) was used as indicated. IBMX (3-Isobutyl-1-methylxanthine; Sigma, #I7018) was used at 10, 50 or 500 μM. PGE2 (Prostaglandin E2; Tocris, #2296) was used at 10 μM. Isoproterenol hydrochloride (Calbiochem, #420355) was used at 10 μM. LRE1 (Sigma, SML1857) was used at 50 μM. KT5720 (Sigma, #K3761) was used at 10 μM. TRULI (gift from the Tri-I TDI) was used at 500 nM. Leptomycin B (Cayman Chemical Company, # 10004976) was used at 46 nM. Cycloheximide (Sigma, # 01810) was used at 20 μg/mL.
Bicarbonate stimulation of human melanoma cells was performed under ambient CO2 conditions at 37°C. Cells were cultured in pH 7.2 stabilized media deficient in bicarbonate for 6 h, and then stimulated by replacing media with regular culture media containing bicarbonate.
Bicarbonate stimulation of human melanoma cells was performed under ambient CO2 conditions at 37°C. Cells were cultured in pH 7.2 stabilized media deficient in bicarbonate for 6 h, and then stimulated by replacing media with regular culture media containing bicarbonate.
8
Regulation of Oxidative Stress Pathways
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Antibodies recognizing BACH1 (F-9), anti-Lamin B2 (C-20), anti-Tubulin (TU-02) and anti-cytokeratin 16 (sc-53255) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). anti-NRF2 (D1Z9C) was obtained from Cell Signaling Technology (Danvers, MA, USA), anti-HMOX1 (ab13243) and anti-cytokeratin 17 (ab109725) were obtained from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies were obtained from Life Technologies (Carlsbad, California, USA). The siRNAs used as control or against BACH1, NRF2 and KEAP1 were the SMART pool: ON-Target Plus from Dharmacon (Lafayette, CO, USA). MG132 was obtained from Santa Cruz Biotechnology, and Leptomycin B was obtained from Cayman Chemicals (Ann Arbor, MI, USA). 9Δ-THC was purchase to Sigma Aldrich (San Louis, MI, USA) and other cannabinoids with a purity higher than 97% were obtained from Prof. Giovanni Appendino (University of Eastern Piedmont, Novara, Italy).
9
Bicarbonate Stimulation of Melanoma Cells
10
Multiplex Protein and RNA Analysis
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Primary antibodies used were anti-UPF1 (Bethyl laboratories, Montgomery, TX, A300-38A, CST, Newburyport, MA, 12040S and Abcam, Cambridge, UK, ab109363), anti-FREM2 (Invitrogen, Carlsbad, CA, PA5-20982), anti-FLAG (Abcam, ab18230), anti-Actin (CST, 4967S), anti-GFAP (Abcam, ab53554), anti-Nestin (Abcam, ab22035) and anti-Beta III tubulin (Abcam, ab18207). Secondary antibodies used include goat anti-rabbit Alexa 488 (Invitrogen, A-11008), goat anti-mouse Alexa 488 (Invitrogen, A-11001), goat anti-rabbit Alexa 594 (Invitrogen, A-11012), goat anti-mouse Alexa 594 (Invitrogen, A-11005), donkey anti-goat Alexa 647 (Invitrogen, A-21447), donkey anti-rabbit Alexa 488 (Invitrogen, A-21206), donkey anti-mouse Alexa 594 (Invitrogen, A-21203). The RNAScope Multiplex Fluorescent V2 Assay (ACD, Newark, CA, 323100) was used with RNAscope Probes include polyA RNA (ACD, 318631) and FREM2 (ACD, 482841). Opal570 (Akoya Biosciences, Marlborough, MA, FP1488001KT) was used for visualization of RNAscope probes. SMARTpool Accell siRNA, which utilizes 4 siRNAs, was used for knockdown of FREM2 (Dharmacon, Lafayette, CO, E-021693-00-0010) and UPF1 (Dharmacon, E-011763-00-0010) according to manufacturer’s instructions. Non-targeting siRNAs used were cat. D-001910-10-20 (Dharmacon). Huh7-Lunets were treated with 60 ng/mL of Leptomycin B (Cayman Chemical) for 16 hours.
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