LADANIA067 is not toxic or proapoptotic in A549 cells.

<p>(A) A549 cells were left untreated or treated with LADANIA067 with the amounts and times as indicated. The impact of LADANIA067 on cell morphology was visualized by an Axiovert 2000 ApoTome microscope (100-fold magnification) and monitored by photography. (B) A549 cells were left untreated or treated with 50 µg/ml, 100 µg/ml or 200 µg/ml LADANIA067 or 1 µM Staurosporine for the indicated time periods. Cell proliferation/viability was estimated via colorimetric assay that followed incubation of cells with MTT. The proliferating cells are depicted, whereby the untreated controls were arbitrarily set as 100%. Data represent ±SD of twelve biological samples. (C) A549 cell were transfected with a constitutive active CMV promoter luciferase plasmid. Eight hours post transfection cells were left untreated or treated with the indicated amounts of LADANIA067 or 10 µg/ml Cycloheximide respectively for 18 hours. Data represent ±SD of nine biological samples and are given as the ratio of luciferase activity relative to the untreated control. (D) A549 cells were incubated in the presence and absence of 50 µg/ml, 100 µg/ml or 200 µg/ml LADANIA for 24 h, 48 h and 72 h. A549 cells that were treated with Staurosporine (1 µg/ml) in addition to EDTA (5 mM) for 4 hours served as control. Percentage of viable cells was measured by staining of dead cells with propidium iodide (PI) as described in <i>Materials and Methods.</i> Data represent ±SD of nine biological samples. Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001) (B, C, D). (E) A549 cells were left untreated or were treated with 1 µM Staurosporine for 8 h or 50 µg/ml or 100 µg/ml LADANIA067 for 24 h or 48 h. Upon stimulation cell lysates were prepared and subjected to Western blot, and caspase mediated PARP cleavage was determined as a measure for apoptosis induction. ERK2 expression was monitored as loading control.</p

LADANIA067 inhibits IAV replication at early times of infection.

<p>(A) A549 cells were left untreated or treated with 100 µg/ml of LADANIA067, which was added at the indicated times during infection with FPV (H7N7) (MOI = 1). (B-D) A549 cells and/or virus were left untreated or treated with 100 µg/ml of LADANIA067 pre-infection or post-infection (p.i.). For infection FPV (H7N7) (MOI = 0.1), SO-IAV (H1N1pan) (MOI = 1) and PR8 (H1N1) (MOI = 5) have been used. Supernatants were assayed for progeny virus particles 8 h p.i. Data represent means ±SD of 4 biological samples (A) or 6 biological samples (B–D). Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001).</p

LADANIA067 inhibits viral internalisation.

<p>(A) A549 cells were left untreated or treated with 100 µg/ml LADANIA067 for 1 h at 37°C. Biotinylated <i>Sambucus nigra</i> agglutinin (SNA) (1 µg/ml) was added and cells were incubated for 1 h at 4°C with A549 cells. Subsequently, cells were incubated with Cy3-conjugated streptavidin to detect lectins for fluorescence microscopy using an Axiovert 2000 ApoTome microscope with AxioCam digital camera and AxioVision software (Zeiss) (20-fold magnification) (B) LADANIA067 was diluted as indicated. Influenza A/FPV/Bratislava/79 was diluted in PBS (4×10⁵ pfu/well) and 50 µl was added per well of a 96-well plate. After preincubation of 45 min, chicken erythrocytes (1∶20 in PBS) were mixed with the solution. In samples where viruses were preincubated with LADANIA067 or CYSTUS052, up to a certain dilution the virus particles are no longer capable of agglutinating erythrocytes, indicating an interaction of LADANIA067 or CYSTUS052 with the viral HA. (C) A549 cells (upper panel) or MDCK cells (lower panel) were left untreated or treated with 200 µg/ml LADANIA067 30 min before infection. Also the influenza virus A/Puerto Rico/8/34 was left untreated or was incubated with the same amount of LADANIA067 at 4°C for 30 min. Subsequently cells were left untreated or infected (MOI = 10) with virus for 30 min at 4°C till a temperature shift to 37°C for further 15 min. Upon stimulation cells were washed twice with PBS and cell lysates were prepared and subjected to Western blot. Viral M1 protein was determined as a measure for attached and internalized virus particles. A549 cells were left untreated or treated with 100 µg/ml LADANIA067 for 4 h prior to stimulation with (D) 5 ng/ml TNFα for 20 min or (E) 30 ng/ml EGF for 10 min. (F) Influenza virus A/Puerto Rico/8/34 was left untreated or was incubated with the indicated amounts of LADANIA067 supplemented with medium for 30 min at 4°C. Subsequently, cells were washed with PBS and incubated for 60 min at 37°C without virus or with virus which was left untreated or was pretreated with LADANIA067. Upon stimulation cell lysates were prepared and subjected to Western blot. (D) Expression of IκBα, (E, F) phosphorylation of EGFR and ERK2 were detected and (D, E, F) EGFR or ERK2 expression was monitored as loading control.</p

LADANIA067 impairs IAV replication.

<p>A549 cells were left untreated or treated with the indicated amounts of LADANIA067 before and during infection with the influenza virus strains A/FPV/Bratislava/79 (FPV) (H7N7) (8 h: MOI = 0.1; 24 h: MOI = 0.01) (A), A/NRW/173/09, an pandemic swine-origin-IAV (SO-IAV) (H1N1pan) (8 h: MOI = 1.5; 24 h: MOI = 0.5) (B) or A/Puerto Rico/8/34 (PR8) (H1N1) (MOI = 0.01) (C). In (C) cells were left untreated or treated with the indicated amounts (left panel) or 50 µg/ml of LADANIA067 before and during infection. (A–C) The virus was pretreated with LADANIA067 as well. Supernatants were assayed for progeny virus particles at the times indicated. Data represent means ±SD of 4 biological samples. Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001).</p

LADANIA067 efficiently blocks influenza virus propagation <i>in vivo</i>.

<p>(A, C) Four 8-week-old BALB/c mice per group were intranasally infected with sublethal doses (A) 10³ pfu or (C) 5×10² pfu of FPV (H7N7). (B) Eight 8-week-old BALB/c mice per group were intranasally infected with sublethal doses of recombinant PR8 (H1N1rec) (4×10² pfu). Mice were either solvent or LADANIA067 treated (1.5 ml per mice, at a concentration of (A, C) 10 mg/ml or (B) 15 mg/ml) via inhalation, twice a day for (A, B) three days or (C) five days. (A, B) Virus titers of infected lungs were determined 3 days p.i. in standard plaque titrations. (C) Body weight of the infected mice was monitored.</p

LADANIA067 shows no tendency to induce virus drug resistance.

<p>A549 cells were infected with FPV (MOI = 0.001) for 24 h and left untreated or treated with 100 µg/ml of LADANIA067 or 5 µM amantatine. Cells and virus were pre-incubated with LADANIA067 for 30 min and LADANIA067 has been added to the medium throughout the infection. Amantadine was added to the medium upon virus inoculation of the cells. The supernatants were taken and employed for infection in the next round of investigation.</p

Impaired NF-κB signalling interferes with spatial pattern separation.

<p>A <i>spatial pattern separation-Barnes Maze (SPS-BM)</i> was developed to test DG dependent spatial pattern separation (<b>a</b>). During consecutive days of training the mice had to find the food house (location F), but only one of the identical seven houses on the plate is freely accessible. Dependent on distal extramaze cues the animals have to find the open food house. The presentation of several identical objects in an environment should lead to overlapping activation in place cells. The analysis of the SPS-BM shows highly significant memory deficits in NF-κB ablated mice (<i>n</i> = 9) as compared to controls (<i>n</i> = 8). Latency (<b>b</b>), distance (<b>d</b>) and error rate (<b>c</b>) were significantly increased in IκB/tTA mice. Reactivation of NF-κB in IκB/tTA animals by doxycyline treatment (<i>n</i> = 13; lower panel) improved the performance considerably. Mice showed significantly decreased latency (<b>e</b>), the error rate (<b>f</b>) and distance covered (<b>g</b>) was comparable to control mice (<i>n</i> = 6). two-way ANOVA evaluation; error bars: SEM; S = Start position; F = food house.</p

NF-κB regulates mossy fiber projections to CA3 and replenishment of the dentate gyrus.

<p>(<b>a</b>) Immunostaining for neurofilament M (NF-M) in control mice (IκB/-) reveals a fasciculated organisation of mossy fibers, connecting granule cells to their target cells in CA3. (<b>b</b>) Impairment of mossy fiber projections after neuronal NF-κB ablation (IκB/tTA). (<b>c</b>) Statistical analysis of NF-M staining shows significantly reduced mossy fiber bundles in IκB/tTA mice (<i>n</i> = 8) as compared to controls (<i>n</i> = 4) (<i>P</i> = 0.0034); (<b>d</b>) Synapses stained for synaptophysin are organised in a laminated fashion. Strongest staining was visible in the stratum lucidum (SL), which corresponds to the termination field of mossy fibers on dendrites of CA3 neurons. (<b>e</b>) In contrast, staining is strongly reduced in the SL after NF-κB ablation, suggesting an important role of NF-κB in regulating synaptic density. (<b>f</b>) NF-κB ablation results also in a significantly decreased dentate gyrus size (white bar in <b>a</b>, <b>b</b>, <i>n</i> = 9) compared to control mice (<i>n</i> = 6) (<i>P</i> = <0.0001). (<b>g</b>, <b>h</b>) Electron microscopy shows that size and frequency of mossy fiber boutons (MFB) are reduced after NF-κB inhibition (blue, mossy fiber boutons; red, dendritic excrescences). (<b>i</b>) The size of MFB is reduced in IκB/tTA mice. (<b>j</b>, <b>k</b>) Scheme of structural defects after NF-κB ablation. Scale bars (a,b) 100 µm, (d,e) 20 µm, (g,h) 2 µm); DG-dentate gyrus, SL-stratum lucidum, PY-pyramidal layer, MFB-mossy fiber boutons. Error bars, SEM. ^**<i>P</i><0.01; ^***<i>P</i><0.0001; Unpaired t-test, two tailed.</p

NF-κB dependent regulation of PKA and FOXO1.

<p><b>A</b> (<b>a</b>, <b>b</b>) Forkhead box protein O1 (FOXO1) is expressed in the DG in control mice (IkB/-), but is downregulated in NF-κB deficient mice (<b>c</b>, <b>d</b>). (<b>e</b>, <b>f</b>) Doxycycline induced reactivation of NF-κB leads to re-expression of the transcription factor Foxo1 as shown by in situ hybridisation (encoded in false colour). <b>B</b> (<b>a</b>, <b>b</b>) The catalytic subunit alpha of Protein kinase A (PKA) is expressed in the DG of control mice (IκB/-), but is strongly downregulated in NF-κB deficient mice (<b>c</b>, <b>d</b>). Reactivation of NF-κB by doxycycline treatment results in re-expression of PKA (<b>e</b>, <b>f</b>); as shown by in situ hybridisation (encoded in false colour, data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030838#pone.0030838-Kaltschmidt2" target="_blank">[19]</a>). <b>C</b> (<b>a</b>, <b>b</b>) Phosphorylated PKA substrates are reduced in granule cells of the dentate gyrus as detected by phospho-specific antibody. (<b>c</b>) Fluorescence intensity is significantly decreased in IκB/tTA mice (<i>n</i> = 4) as compared to controls (<i>n</i> = 4) (<i>P</i> = <0.0001) (<b>d</b>) Phosphorylation of LKB1, a substrate of PKA, is reduced in hippocampal brain extracts from IκB/tTA mice as detected by Western blotting. Scale bar (a, b) 20 µm; Error bars: SEM; DG dentate gyrus.</p

Reactivation of NF-κB leads to regrowing of the dentate gyrus.

<p>(<b>A</b>) (<b>a</b>) The size of the dentate gyrus (DG) in NF-κB ablated mice is strongly reduced. Reactivation of NF-κB leads to increased number of granule cells within the DG (<b>b</b>). (<b>B</b>) Statistical analysis shows a significant decrease of granule cells after NF-κB ablation (IκB/tTA mice, <i>n</i> = 9; as compared to control mice IκB/-; <i>n</i> = 3, (<i>P</i> = <0.0001)). Note, after reactivation of NF-κB by doxycycline treatment the number of granule cells is increased in IκB/tTA mice (<i>n</i> = 7) (<i>p</i> = 0.0002). No difference in the control groups IκB/- (<i>n</i> = 3) and IκB/- +DOX (<i>n</i> = 3) was detected (<i>p</i> = 0.0605). (<b>C</b>) Doublecortin (DCX) staining marking neuronal precursors. After doxycycline treatment the number of DCX positive cells is strongly decreased in IκB/tTA mice (<i>n</i> = 3) (<b>c</b>, <b>d</b>) as compared to NF-κB ablated group (<i>n</i> = 3) (<b>a</b>, <b>b</b>). (<b>D</b>) Statistical evaluation shows significantly less DCX positive cells after doxycycline treatment in IκB/tTA mice (<i>n</i> = 3) as compared to untreated IκB/tTA group (<i>n</i> = 3) (<i>p</i> = <0.0001). No difference in control groups IκB/- (<i>n</i> = 3) and IκB/- +DOX (<i>n</i> = 3) was detected (<i>p</i> = 0.1063). (<b>E</b>) Statistical analysis of neurofilament-M (NF-M) staining shows significantly reduced mossy fiber bundles in IκB/tTA (<i>n</i> = 8) mice as compared to controls (<i>n</i> = 4) (<i>p</i> = 0.0034). Reactivation of NF-κB results in mossy fiber outgrowth of newborn neurons, thus increasing the area of mossy fibers in doxycycline treated IκB/tTA mice as compared to the untreated group (<i>p</i> = 0.0024, t-test with Welch's correction). No difference in the control groups IκB/- (<i>n</i> = 4) and IκB/- +DOX (<i>n</i> = 3) was detected ( <i>p</i> = 0.1897). Error bars: SEM; <i>p</i><0.05; ^**<i>p</i><0.01; ^***<i>p</i><0.0001.</p