Leptomycin b lmb

96-well flat-bottom plates (Costar) were seeded with 1.5 x 10⁴ A549 cells/well and incubated overnight at 37°C/5%CO₂. Clopidogrel and Triamterene were prepared in DMSO to a stock concentration of 10 mM. Drugs plates were prepared using the D300 BioPrinter digital drug dispenser (HP, Palo Alto, CA) and dispensed into 96-well plates containing virus infection media containing MEM supplemented with 0.3% BSA and TPCK-treated trypsin (1 ug/ml) (Worthington, Lakewood, New Jersey) to the final concentrations 500, 200, 150, or 100 uM. A/WSN/33 virus (MOI = 0.01) was prepared in infection media. A549 cell plates were washed 2x with PBS. Equal volumes of infection and drug dilutions were transferred to the A549 cells (excluding control wells) to infect cells (MOI = 0.01) and achieve final concentrations of 250, 100, 75, or 50 uM of the drug. The wells were normalized to 1% DMSO. The plates were incubated for 12h or 24h at 37°C/5% CO₂. Following incubation, the supernatants were removed, the plates were washed 2x with PBS, and the cells fixed with acetone:methanol (20:80; Sigma). Leptomycin B (LMB) (Sigma, St. Louis, MO) was used as the positive control for inhibition of influenza replication and was administer on cells 2h prior to infection [58 (link), 59 (link)].

For the treatment of melanoma cells with inhibitors, 2×10⁵ melanoma cells per well were seeded in six-well plates. After 4 h, the cells were treated with 20 mM of the PI3 kinase inhibitor LY-294002, 20 mM of Wortmannin (both Sigma-Aldrich, St Louis, MO, USA) or a control substance for 16 h. For the inhibition of XPO1, cells were incubated with 10 ng/ml of leptomycin B (LMB, Sigma-Aldrich) for 20 h. For MEK inhibition, cells were treated with 10 μM PD98059 or 10 μM U0126 (Merck Millipore, Billerica, MA, USA) for 48 h. RNA polymerase was inhibited using 5 μM α-amanitin (AppliChem, Darmstadt, Germany). Treatment with MG132 (Merck Millipore) at final concentrations of 5, 10 and 20 μM for 24 hours was used to inhibit the proteasome. For protein kinase inhibition, cells were treated with 100 μM staurosporine (Merck Millipore) for 16 hours.

RVH-421 (ACC 127) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures, and SK-MEL-5 (HTB-70) was obtained from ATCC as part of the NCI-60 cancer cell line panel. Each cell line was cultured in RPMI 1640 medium (Corning, 10–040-CV) supplemented with 10% FBS and 1% penicillin and streptomycin. Cancer cells were seeded at a concentration of 3 × 10⁵ cells into 12-well plates and incubated at 37°C overnight. Cells were washed with warm Dulbecco's PBS before being treated with 10 μM KPT-335 (Selleckchem, S7707) or 5 ng/ml leptomycin B (LMB; Sigma, L2913) in the presence or absence of 50 ng/ml IFN-γ (PeproTech, 300–02) in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin for 32 h.

HeLa, 293T and U373MG cells were cultured in DMEM supplemented with 10% Fetal Calf serum (FBS). BHK-U3GFP indicator FAG (Fluorescence Activated GFP) cells were cultured in DMEM supplemented with 5% FBS and 500 µg/mL G418 (Gibco). Leptomycin B (LMB) (Sigma) was added to culture medium to a final concentration of 10 nM for 4 h. Hypoxic conditions (2% O₂, 5%CO₂ and 93% N₂) were induced by a continuous flow of nitrogen using a Forma Series II Water Jacket CO2 incubator (model: 3131; Thermo Scientific).

HeLa cells expressing endogenous STAT1 and STAT1-negative U3A cells [26] (link) were cultured at 37°C in a humidified 5% CO₂ atmosphere in Quantum 101 medium and Dulbecco’s modified Eagle’s medium (both obtained from PAA Laboratories), respectively. For both human cell lines, media were supplemented with 10% foetal calf serum (FCS; Biochrom), 1% penicillin, and 1% streptomycin. Transfection was achieved with MegaTran1.0 (Origene) according to the manufacturer’s recommendation. Twenty-four hours after transfection, cells were either left unstimulated or stimulated with 5 ng/ml recombinant human IFNγ (Biomol). Subsequently, cells were incubated with either 500 nM staurosporine (Sigma-Aldrich) or a combination of 0.8 mM sodium vanadate and 0.2 mM H₂O₂ for the time periods indicated. The anti-fungal antibiotic leptomycin B (LMB, Sigma-Aldrich) was used at a final concentration of 10 ng/ml to block CRM1-mediated export.

Primary human dermal fibroblasts from patients with HGPS and healthy donors (see Table 1) were cultured in MEM (Invitrogen) supplemented with 15% FBS (Invitrogen), nonessential amino acids, and antibiotics. All experiments were carried out with fibroblast cultures at passage numbers 12–16. Where indicated, fibroblasts were treated with 1 nM leptomycin B (LMB; Sigma‐Aldrich) for 1, 3, or 6 days diluted in ethanol to a final concentration of 0.1% in the culture medium, or with 25 µM of the farnesyl transferase inhibitor (FTI) lonafarnib (Sigma‐Aldrich) for three days. Fibroblasts were transfected with LLipofectamine">ipofectamine 3,000 following manufacturer's protocol (Invitrogen). Stably transfected fibroblasts were obtained by culturing them for 12 days with 200 μg/ml G418 (Invitrogen). HeLa cells were double‐transfected with pFlag‐CRM1 vector and vector expressing either a short hairpin RNA (shRNA) specific for the human nuclear transport factor 2 gene (NUTF2) or a scrambled shRNA control (GeneCopoeia, Inc.), using LLipofectamine">ipofectamine 2000 (Invitrogen). Stably transfected HeLa cells were obtained by culturing them for 6 days in the presence of 1 μg/ml puromycin and 800 μg/ml G418. Cloning strategies to obtain vectors pEGFP‐C1‐LB1, pFLAG‐CRM1, and pCRM1 promoter are provided under request.