Journal: PLoS ONE

Article Title: Inflammatory response of mesenchymal stromal cells after in vivo exposure with selected trauma-related factors and polytrauma serum

doi: 10.1371/journal.pone.0216862

Figure Lengend Snippet: (A) Analysis of cytokines in serum from healthy volunteers (control; n = 5), in AB-serum (n = 1) and in serum from polytrauma (PT) patients (n = 5). Mean and SD are shown. Cytokine levels in serum from PT patients were compared to cytokine levels in serum from healthy volunteers. Statistical significance was assessed via student's t-test (with correction for multiple comparisons), * P < 0.05. IL6: interleukin 6; IL10: interleukin 10; CXCL1: C-X-C motif chemokine ligand 1; CCL2: C-C motif chemokine ligand 2. (B) Effect of serum from PT patients on proliferation of MSC. Three MSC preparations and five individual sera from PT patients were used (randomized combination). Serum was collected from PT patients 0/4/12/24/48/120/240 h after admission to hospital. MSC cell count was determined by fluorescence intensity and compared to cell growth in Alpha-MEM without (negative control) or with supplement (positive control).

Article Snippet: The PTCH consisted of 10 ng/ml IL1B (PeproTech, Hamburg, Germany), 10 ng/ml IL6 (BIOMOL, Hamburg, Germany), 500 ng/ml C3a, 100 ng/ml C5a (both Calbiochem, Darmstadt, Germany), 60 ng/ml HMGB1, 300 ng/ml THBD, 450 pg/ml THPO.

Techniques: Control, Cell Counting, Fluorescence, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Inflammatory response of mesenchymal stromal cells after in vivo exposure with selected trauma-related factors and polytrauma serum

doi: 10.1371/journal.pone.0216862

Figure Lengend Snippet: Left column: Cells were cultured in medium supplemented with 20% AB-serum with or without (-, three biological replicates, 2 technical replicates) addition of trauma factors (interleukin 1 beta (IL1B, n = 3), polytrauma cocktail high (PTCH, n = 3), polytrauma cocktail low (PTCL, n = 3)) for 6 h or 24 h. Statistical significance was assessed via two-way ANOVA (with correction for multiple comparisons). Right column: cells were cultured in medium supplemented with 20% AB-serum (three biological replicates, 2 technical replicates) or serum from polytrauma patients (PTS) taken at certain times (0 h, 4 h, 12 h, 24 h, 48 h, 120 h, 240 h; three different MSC preparations and sera from five different patients were used for each point of time) for 6 h. Cytokine levels of cell culture supernatant from PTS-treated MSC were compared to cytokine levels of cell culture supernatant from AB-serum-treated MSC. Statistical significance was assessed via student's t-test (with correction for multiple comparisons), * P < 0.05. IL6: interleukin 6; IL10: interleukin 10; VEGFA: vascular endothelial growth factor-A; CXCL1: C-X-C motif chemokine ligand 1; CCL2: C-C motif chemokine ligand 2.

Article Snippet: The PTCH consisted of 10 ng/ml IL1B (PeproTech, Hamburg, Germany), 10 ng/ml IL6 (BIOMOL, Hamburg, Germany), 500 ng/ml C3a, 100 ng/ml C5a (both Calbiochem, Darmstadt, Germany), 60 ng/ml HMGB1, 300 ng/ml THBD, 450 pg/ml THPO.

Techniques: Cell Culture

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