Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Ankle1 shuttles between nucleus and cytoplasm. a Schematic representation of Ankle1’s domain organization depicting predicted ankyrin repeats, the LEM domain and a GIY-YIG nuclease domain. Putative nuclear export sequences (NES1, NES2) and nuclear localization sequences (NLS1, NLS2), identified in silico are indicated. b Immuno-fluorescence analysis of ectopic Ankle1-V5 in U2OS cells without or following a 3 h treatment with 50 nM leptomycin B, an inhibitor of CRM1-mediated export. Cells were stained with antibodies to V5, and DNA with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: In Silico, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Localization of Ankle1 fragments containing different domains and export and import signals. Localization of GFP-tagged Ankle1 truncation constructs ectopically expressed in U2OS ( a ) and HeLa ( b ) cells was determined by confocal fluorescence microscopy. Molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. Cells were fixed after 3 h of mock or leptomycin B treatment. DNA was counterstained with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Construct, Fluorescence, Microscopy

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Mutation analyses identify NES2 and NLS2 as the predominant sequences controlling nucleo-cytoplasmic shuttling of Ankle1. a , b , d , e U2OS cells were transiently transfected with Ankle1-V5 carrying point mutations in NLS or NES sequences and either mock-treated or treated with leptomycin B for 3 h and processed for confocal immunofluorescence analyses using antibodies to V5 and DAPI to detect DNA. Scale bars: 10 μm. c Mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type Ankle1-V5, Ankle1-NES1mut-V5 or Ankle1-NES2mut-V5 were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. Data were obtained from three independent experiments and analyzed using Student’s t -test. Ankle1-NES1, P = 0.002; Ankle1-NES2, P = 5.4E-21; n = 50; 15–17 cells each from three independent experiments

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Expressing

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY and FKH1 cells were treated with ( A ) LMB (20 nM, 3 h) or ( C ) KPT-185 (1 μM, 24 h) and NUP214 (green) and CRM1 (magenta) localization was studied by confocal microscopy. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm. Quantitative analysis of the number of NUP214 nuclear bodies in ( B ) LOUCY and ( D ) FKH-1 cells. At least 150 cells were analyzed for each condition. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Confocal Microscopy, Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY cells (SET-NUP214) were treated with ( A ) LMB (20 nM, 3 h) or ( B ) KPT-185 (1 μM, 24 h). Cells were allowed to grow in drug-free medium for up to 48 h, after clearance from the respective drug. The presence of SET- NUP214 nuclear bodies was evaluated by immunofluorescence using anti-NUP214 (green) and anti-CRM1 (magenta) antibodies. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY cells (SET-NUP214) were treated with ( A ) LMB (20 nM, 3 h) or ( B ) KPT-185 (1 μM, 24 h) and cells were allowed to grow in drug-free medium for up to 48 h after treatment. The formation of SET-NUP214 (green) nuclear foci is accompanied by the accumulation of CRM1 (magenta) in these structures. Shown are representative confocal images. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: Leukemia cell lines LOUCY ( A ), MEGAL ( B ), and FKH-1 ( C ) were treated with LMB (20 nM) or KPT-185 (1 μM) for the indicated time points and cell viability was measured by Trypan Blue exclusion dye. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques:

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LMB or KPT-185 treated LOUCY, MEGAL, and FKH-1 cells were allowed to grow in drug-free medium for up to 48 h after drug clearance. ( A ) Cell viability was determined by Trypan Blue exclusion dye. ( B ) WST-1 assay was performed to measure cellular metabolic activity. Absorbance was measured at 450 nm. Results are normalized to untreated cells and expressed as percentage. ( C ) Colony forming assays of LMB and KPT-185 treated MEGAL and FKH-1 cells. Colonies were visualized under a 10× microscope objective after growth in drug-free Methocult™ medium for 14 days. Scale bars, 100 μm. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: WST-1 Assay, Activity Assay, Microscopy

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: Flowcytometric analysis of LOUCY, MEGAL, and FKH-1 cells after withdrawal from LMB or KPT-185 treatment. Cell proliferation was evaluated by using FITC-Ki-67 antibodies. ( A ) Histogram representation and ( B ) quantification of FITC-Ki-67 positive cells in the population of single cells at the indicated time points.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: