Journal: Thorax

Article Title: Fas activation alters tight junction proteins in acute lung injury.

doi: 10.1136/thoraxjnl-2018-211535

Figure Lengend Snippet: Wild-type (WT) and Fas deficient (lpr) mice were treated with one intratracheal instillation of recombinant human soluble FasL (rh-sFasL, 25 ng/g body wt), or PBS (as control). At 16 h post-instillation, we measured (A) the number of nuclei containing DNA strand breaks (TUNEL-positive signal) in lung tissue sections, and (B) caspase-3 activity in the lung homogenates. Merged images of representative lung tissue sections from PBS or FasL-treated WT mice (C), showed that occludin and ZO1 protein signals (red) were diminished in the alveolar walls only in areas with TUNEL positive cells (green). Original image magnification X400. n of 5 mice per group for the immunofluorescence analyses. In the graphs, each dot represents an individual mouse (n= 10 mice per group). Horizontal bars represent means. *P < 0.05 vs WT-PBS group

Article Snippet: The irreversible caspase-3 inhibitor zDEVD.FMK, and the negative control for caspase inhibitors zFA.FMK were purchased from BD Pharmingen.

Techniques: Recombinant, TUNEL Assay, Activity Assay, Immunofluorescence

Journal: Thorax

Article Title: Fas activation alters tight junction proteins in acute lung injury.

doi: 10.1136/thoraxjnl-2018-211535

Figure Lengend Snippet: HPAEpiC monolayers were pre-incubated with caspase-3 inhibitor zDEVD.fmk (or zFA.fmk or vehicle as controls) and exposed for 2 h to rh-sFasL (100 ng/mL or 300 ng/mL) or medium only (MO) (A-C). After treatment, we measured (A) caspase 3-activity, (B) percentage of cell death and (C) FITC-albumin permeability in these cell monolayers. HPAEpiC monolayers were also pre-incubated with the tyrosine-kinase specific inhibitor genistein (or vehicle as control) (D-H) and exposed for 2 h to rh-sFasL (100 ng/mL or 300 ng/mL) or medium only (MO). After treatment, we measured IL-8 concentration in cell supernatant by ELISA (D), percentage of cell death by PrestoBlue (E), permeability to FITC-albumin (F), IL-6 concentration in cell supernatant by ELISA (G), and tyrosine kinase activity in the cell monolayers (H). Cell monolayers treated with medium only were used to determine 100% survival. Results from 4 separate experiments performed in duplicate. Each dot of the graphs represents a single data point. Horizontal bars represent means. *P<0.05 vs all conditions with MO; # P< 0.05.

Article Snippet: The irreversible caspase-3 inhibitor zDEVD.FMK, and the negative control for caspase inhibitors zFA.FMK were purchased from BD Pharmingen.

Techniques: Incubation, Activity Assay, Permeability, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Thorax

Article Title: Fas activation alters tight junction proteins in acute lung injury.

doi: 10.1136/thoraxjnl-2018-211535

Figure Lengend Snippet: After 2 h exposure of rh-sFasL (+FasL) at the dose of 100 ng/mL or medium only (-FasL), the concentrations of occludin and ZO1 proteins were measured by ELISA in HAECpiC monolayers pre-incubated with the caspase-3 inhibitor zDEVD.fmk, its inactive analog (zFA.fmk), or the tyrosine kinase inhibitor genistein or vehicle (DMSO). Compared to control cells, rh-sFasL decreased the concentrations of occludin and ZO1 in cell protein extracts (A and B, respectively). Representative immunofluorescence images (original image magnification X400) (C) show the expression of occludin and ZO1 proteins (red signals) localized along the cytoplasmic membrane of control cells in HAECpiC monolayers (−rhFasL/+vehicle). Exposure to rh-sFasL resulted in a global decrease of occludin and ZO1 fluorescence signals that was particularly profound in the cytoplasmic extensions, while some signal remained close to the cell nuclei (DAPI staining, blue signal). Only pre-incubation with the caspase-3 inhibitor zDEVD.fmk prevented the sFasL-induced decrease of occludin and ZO1 proteins evaluated both by ELISA (A and B) and by immunocytochemistry (C). Representative immunofluorescence images at larger magnification (x1000) show the decreased expression of occludin and ZO1 proteins (red signals) in HAECpiC monolayers treated with rh-sFasL (+rh-sFasL/+vehicle) compared with control cells (−rh-sFasL/+vehicle) (D) Results from 4 separate experiments performed in duplicate. Each dot of the graphs represents a single data point. Horizontal bars represent means. *P<0.05 vs their corresponding-rh-sFasL conditions.

Article Snippet: The irreversible caspase-3 inhibitor zDEVD.FMK, and the negative control for caspase inhibitors zFA.FMK were purchased from BD Pharmingen.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Immunofluorescence, Expressing, Fluorescence, Staining, Immunocytochemistry

Journal:

Article Title: The CB1/VR1 agonist arvanil induces apoptosis through an FADD/caspase-8-dependent pathway

doi: 10.1038/sj.bjp.0705532

Figure Lengend Snippet: Schematic diagram highlighting the proposed mechanism for arvanil-induced apoptosis. Arvanil induces FADD clustering, which allows the recruitment of caspase-8. Then, this caspase is cleaved and activated, leading to an apoptotic cascade that includes Bid truncation, leaking of cytochrome c from mitochondria and activation of different effector caspases (-3, -7, -9). To complement this pathway, arvanil induces ROS that may help to DISC formation and may also induce the mitochondrial release of cytochrome c.

Article Snippet: The cell-permeable inhibitors of caspases zDEVD-fmk and zIETD-fmk ( N -benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone and N -benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone, respectively) were from Bachem (Bubendorf, Switzerland).

Techniques: Activation Assay