Elevated Bfl-1 expression upon allergen provocation in birch-pollen allergic patients (A) and in lesions of atopic dermatitis (AD) and psoriasis patients (PSO) (B).

<p>An enzymatical staining for tryptase followed by an immunohistochemical staining of Bfl-1 was performed. The results are presented as the percentage of mast cells expressing Bfl-1.</p

Bfl-1 is expressed in skin tissue mast cells.

<p>An enzymatical staining for tryptase followed by an immunohistochemical staining of Bfl-1 (upper panel) and was performed on atopic dermatitis (AD) lesional skin. Isotype control staining following tryptase staining (lower panel).</p

siRNA targeting <i>BFL-1</i> but not <i>MCL-1</i> diminishes activation-induced survival of human mast cells.

<p>(A) The upregulation of <i>BFL-1</i> and <i>MCL-1</i> following IgECL in CBMCs is abolished following targeted siRNA treatment as verified 30 hours post-transfection by quantative PCR. Cells were transfected with 100 nM siRNA, sensitized with 1 μg/mL of IgE and challenged with 2 μg/mL anti-IgE before expression was determined. Data correspond to one representative experiment using one donor. Similar result was obtained for another donor. (B) <i>BFL-1</i> but not <i>MCL-1</i> siRNA treated CBMCs show decreased survival upon IgECL compared to control siRNA treated cells. Cells were transfected, sensitized with 1 μg/mL of IgE and 24 hours post-transfection cytokine-deprived and challenged with 2 μg/mL anti-IgE before being enumerated 24 hours later using the vital dye trypan blue. N=6. (C) The upregulation of <i>BFL-1</i> and <i>MCL-1</i> following IgECL in LAD-2 cells is abolished following targeted siRNA treatment as described above. (D) <i>BFL-1</i> but not <i>MCL-1</i> siRNA treated LAD-2 cells show decreased survival upon IgECL compared to control siRNA treated cells. N=4. Change in viability is percentage viable cytokine-deprived cells deducted from viable cytokine-deprived cells following IgECL. (E) Bfl-1 is down-regulated in LAD-2 cells following siRNA treatment targeting <i>BFL-1</i> as compared to control siRNA in immunohistochemical stainings for Bfl-1 expression.</p

Activation-induced mast cell survival following IgECL in presence of ABT-737 and roscovitine.

<p>(A) CBMC treated with 0.5 μM ABT-737 alone or in combination with 5 μM roscovitine following IgECL. N=6–3. (B) LAD-2 treated with 0.5 μM ABT-737 alone or in combination with 5 μM roscovitine following IgECL. N=3 Viability was assessed after 24 hrs using propidium iodide plus FITC-conjugated Annexin V. Change in viability is expressed as percentage viable cells after treatment deducted from untreated cells. ABT-737 = ABT, roscovitine = rosc.</p

IgECL-induced survival of human mast cells.

<p>(A). CBMC upon IgECL. (B) LAD-2 cells upon IgECL. Cells were sensitized with 1 μg/mL of IgE overnight before cytokine-deprived and challenged with anti-IgE. After 24 hrs the cell viability was enumerated using trypan blue exclusion. N=3–4.</p

Casp8 ablation provides neuroprotection in organotypic brain slice cultures.

<p>The coronal brain slices derived from the CRE3 control line and N<i>casp8</i>^−/− homozygous mice were maintained in <i>in vitro</i> conditions for 3 (<b>A1–M</b>) and 14 days (<b>N–X</b>). The presented results were obtained from untreated (<b>A1–D3, I, K, N–X</b>) and TRAIL-treated (<b>E–H, J, L</b>) cultures. Immunohistochemical stainings are depicted in cortical and subcortical regions of CRE3 (<b>A1, A2, E, F, Q, S, W</b>) and N<i>casp8</i>^−/− (<b>B1–D3, G–P, R, T, U, X</b>) brain slices. Examples of single marker (<b>M–W</b>) and double (<b>A1–L, X</b>) immunostainings are presented for S100 (<b>M, N</b>), NeuN (<b>O–T</b>), and β3-tubulin (<b>U, W</b>) as well as for double labeled MAP2 (DAB-brown) and cleaved caspase 3 (SG-black; <b>A1–B2</b>) or GFAP (DAB; <b>D1–D3</b>), TUNEL (DAB; <b>E–H</b>) with MAP2/NeuN antibody cocktail (SG) and S100 (DAB; <b>I–J</b>); IbA1 (DAB; <b>C1–C2</b>) or GFAP (DAB; <b>D1–D3</b>) with NeuN (SG) immunostaining. Scale bar indicates ∼1000 µm on macro images and ∼100 µm on high magnification images of the slices. Abbreviations: <b>DG</b> = Dental Gyrus; <b>CA2; CA4</b> = sectors of hippocampus; <b>TH</b> = Thalamus; <b>AMG</b> = Amygdala; <b>PC</b> = Piriformis Cortex; <b>WM</b> = White Matter.</p

Assessment of lesion volume, cell death and immunoreactivity for MAP2, NeuN, and phospho-tau in control and Ncasp8^−/− brains after CCI.

<p>(<b>A1–A4</b>) Masson's trichrome–stained brain sections were digitalized using the Aperio scanning system. Digitized virtual slides were annotated by encircling the brain tissue loss (cavity) and lesion edges (penumbra) between intact and pathologically changed brain tissue of the control (<b>A1, A3</b>) and N<i>casp8</i>^−/− (<b>A2, A4</b>) mice. Scale bar = ∼1000 µm on low and ∼100 µm on high magnification images. Examples of nuclear NeuN (<b>B–E</b>) and the perikaryonal and dendritic MAP2 (<b>F1–F4</b>) immunostaining are presented in the coronal brain sections (<b>F1, F2</b>), cortical lesions and adjacent structures (<b>B–E, F3, F4</b>) of the control (<b>B, C, F1, F3</b>) and N<i>casp8</i>^−/− (<b>D, E, F2, F4</b>) mice after CCI. Scale bar = ∼1000 µm on low (<b>F1, F2</b>) and ∼100 µm on higher magnification images (<b>B–E, F3, F4</b>). Examples of phospho-tau immunostaining in control (<b>G, H</b>) and N<i>casp8</i>^−/− (<b>I, J</b>) brains are provided at low and high-power magnifications (scale bar = ∼100 µm). Using Aperio software, percentage (<b>K1, L1</b>) and density (n/mm²) (<b>K2, L2</b>) of degenerating (<b>K1,2</b>) and apoptotic (<b>L1,2</b>) neurons was identified by Masson's trichrome stain and cleaved caspase 3 immunostaining, respectively (mean±SEM; n = 8–10 per group and per time point) in lesion areas of brains from CRE3 (white bar) and N<i>casp8</i>^−/− (black bar) mice at 2 h, 6 h, 24 h, 48 h, and 3 weeks after trauma (<b>K1</b>: *<i>p</i> = <.0001; **<i>p</i> = 0.003; ***<i>p</i> = 0.02, <b>K2</b>: *<i>p</i> = 0.0001; **<i>p</i><.0001; ***<i>p</i> = 0.05; ****<i>p</i> = 0.01; <b>L1</b>: *<i>p</i> = 0.02; L2: *<i>p</i> = 0.02; **<i>p</i> = 0.03). (<b>M</b>) SDS-PAGE immunoblot analysis of caspase 3 and cleaved PARP protein levels in mouse brain tissues. Brain samples from impact lesion and corresponding region in the contralateral hemisphere (CLH) of N<i>casp8</i>^−/− mice (lanes 3, 6) were compared with those from CRE3 (lanes 1, 4) and <i>casp8^fl/fl</i> (lanes 2, 5) animals. Lysates were normalized for total protein content (30 µg/lane) and analyzed by SDS-PAGE/immunoblotting using antibodies specific for caspase 3, cleaved PARP, or β-actin, using a multiple antigen detection method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone.0024341-Krajewski1" target="_blank">[39]</a>. (<b>N–X</b>) Examples are provided of phospho-c-Jun immunostaining in ipsilateral semi-hemispheres of brains from the control (<b>N–R</b>) and N<i>casp8</i>^−/− (<b>S–X</b>) mice (magnification scale bar = ∼100 µm). Antibody detection was accomplished using polymer-based EnVision-HRP-enzyme conjugate (DakoCytomation) and diaminobenzidine (DAB) chromogen (brown); nuclei were counterstained with hematoxylin (blue). Percentage (<b>Y</b>) and density (<b>Z</b>) of phospho-c-Jun immunopositive nuclei were determined in impact lesions from control (white bar; Ctrl) and N<i>casp8</i>^−/− (black bar) animals at 6, 24, 48 h, and 21 days post CCI (mean±SEM; n = 8–10 mice per group and per time point) (<b>Y</b>: *<i>p</i> = 0.03; <b>Z</b>: *<i>p</i> = 0.02; **<i>p</i> = 0.05).</p

Caspase 8 gene ablation provides neuroprotection in vitro in primary neuronal cultures and brain organotypic cultures.

<p>(<b>A–B</b>) 3-day-old PNC derived from embryos (E16.5) of CRE3 (<b>A</b>) and N<i>casp8</i>^−/− homozygous mice (<b>B</b>) were treated with recombinant murine TNFα and cycloheximide (CHX), then fixed and stained with Hoechst dye and immunostained with an antibody to NeuN followed by application of Alexa Fluor 594 conjugated anti-mouse antibody. (<b>C1–J4</b>). Untreated (UNT <b>C1–F4</b>), STS-treated, (<b>G1–H4</b>) and recombinant murine TNFα and CHX-treated (<b>I1–J4</b>) 14-day-old neuronal cultures derived from CRE3 and N<i>casp8</i>^−/− mice were preincubated with pSIVA reagent (green) for 15 min prior to Z-fix fixation, then immunostained for MAP2 (red) and stained with DAPI (blue). To assess neuronal phenotype, the transmission light, single (<b>C1, D1, E4, G1, H1, H3, I1, I2, I4, J1</b>), double (<b>C2, D2, E1, E2, F1, F3, G4, H2, H4, I3, J2</b>), and triple (<b>E3, F2, F4, G2, G3, J3, J4</b>) channel images are provided. Colocalization of neuronal marker MAP2 (red) with pSIVA signal (green) resulted in yellow color denoting degenerating neurons whose number was counted and plotted as ratio to the total number of neurons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone-0024341-g002" target="_blank"><b>Figure 2B</b></a>). Single and multichannel panels confirm specificity of the double labeling. PSIVA was visualized from the early (membranous staining) to later stages (staining in the cell body and nucleus, the latter in cells with loss of plasma cell integrity) of neuronal degeneration.</p

Analysis of TUNEL assay and protein biomarkers in cortical and basal ganglia regions of untreated 14-day-old organotypic brain slice cultures (BOCSC).

<p>Statistical analyses of control <b>CRE3</b><i>vs</i><b>N</b><b><i>casp8</i></b><b>^−/−</b> mouse specimens were performed using Student's t-test. The resulting average percentages of positive/immunopositive cells and standard error of mean (SEM) values are provided. <i>P</i> values<0.05 are reported as statistically significant. “NS” stands for nonsignificant (<i>p</i>≥0.05).</p

Caspase 8 deficiency affects functional and histopathological outcome following TBI and facilitates learning and memory retention in aged mice.

<p>(<b>A</b>) Sensorimotor function was measured as the number of foot slips while traversing the length of the beam three times in the beam walking trial. Training was followed by probe 1 (one week interval) before CCI procedure and repeated 7 and 21 days after the procedure (probe 2 and 3). <i>P</i> values result from Student's t-test (*<i>p</i> = 0.009; **<i>p</i> = 0.04). (<b>B</b>) Wire grip scores were quantified using a 5-point scale <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone.0024341-Hall1" target="_blank">[33]</a> to assess motor function. Tests were conducted 1 day before CCI (probe 1) and 7 (probe 2; *<i>p</i> = 0.03) and 21 days (probe 3) after brain trauma. (<b>C</b>) Morris water maze (MWM) performance was tested in the control (Ctrl; n = 12) and N<i>casp8</i>^−/− (n = 15) mice. Average latency to reach and climb the platform or enter the platform zone before and after CCI is shown (mean±SEM) (*<i>p</i> = 0.005; **<i>p</i> = 0.001; ***<i>p</i> = 0.009). Beam walking (<b>D</b>) and wire grip (<b>E</b>) tests were conducted in uninjured 18 month old <i>casp8^fl/fl</i> (Ctrl; n = 19) and N<i>casp8</i>^−/− (n = 20) mice (3 testing sessions, spaced one week apart). <i>P</i> values result from Student's t-test (*<i>p</i> = 0.01). (<b>F</b>) MWM task was performed in the same aged cohort to test memory retention up to 60 days after completion of the learning sessions. Average latency to reach and climb the platform is shown (mean±SEM) (*<i>p</i> = 0.04). (<b>G–I</b>) Histopathological outcomes were compared between the control (Ctrl) and N<i>casp8</i>^−/− mice 2 h, 6 h, 24 h, 48 h, and 3 weeks after the trauma (8–10 mice per time point in each experimental group). Using Aperio scanning system, lesion (<b>G</b>) and cavity (<b>H</b>) volumes were determined by summing each lesion or cavity area multiplied by the distance between each coronal slice (mean±SEM). Plots depict average lesion volume (<b>G</b>: *<i>p</i> = 0.03; **<i>p</i> = 0.002; ***<i>p</i> = 0.003), cavity volume (<b>H</b>: *<i>p</i> = 0.02; **<i>p</i> = 0.03), and neuropathological scores (<b>I</b>: *<i>p</i> = 0.002; **<i>p</i> = 0.02; ***<i>p</i> = 0.004). (<b>J</b>) Time course for density (n/mm²) of NeuN-immunopositive neurons in traumatic brain lesions is depicted for the control (Ctrl) and N<i>casp8</i>^−/− mice. (<b>K</b>) The NeuN immunostaining results are compared among the six experimental groups [control (Ctrl) and N<i>casp8</i>^−/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice] in impact lesions (CCI-treated) or corresponding regions (naïve or sham-operated) at 21 day after procedure. Density (n/mm²) of NeuN-immunopositive neurons in impact lesions or matching ipsilateral regions (IL) and corresponding contralateral (CL) regions of the control (Ctrl) (<b>L</b>) and N<i>casp8</i>^−/− (<b>M</b>) naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice is presented in a time-course manner (<b>L, M</b>) or at 21 day after procedure (<b>N</b>) (<b>J</b>: *<i>p</i> = 0.02; **<i>p</i> = 0.01; ***<i>p</i> = 0.0004; <b>L</b>: *<i>p</i> = 0.0005; **<i>p</i><.0001; ***<i>p</i> = 0.0008; <b>M</b>: *<i>p</i> = 0.0002; **<i>p</i> = 0.006; ***<i>p</i> = 0.002; ****<i>p</i> = 0.0007). (<b>O–P</b>) Percentages of MAP2 immunopositive neurons were compared in impact lesions or corresponding ipsilateral regions of the control (Ctrl) and N<i>casp8</i>^−/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice (8–10 mice/time point/group) and presented as time-course data (<b>O</b>) at 5 time points post CCI (mean±SEM) (<b>L</b>: *<i>p</i> = 0.002) and at 21 day after CCI (bar graph)(<b>P</b>). (<b>Q</b>) Phospho-tau immunoreactivity was assessed in ipsilateral semihemispheres of the control and N<i>casp8</i>^−/− mice 48 h and 3 weeks after CCI (n = 8–10 per group) (mean ± SEM *<i>p</i> = 0.001).</p