ADAM17 depletion in hVSMCs regulates FAS shedding and increases sensitisation to TIMP-3-dependent apoptosis.

<p>A. Western blot for ADAM17 (A17) or FAS in hVSMCs (Cont), infected with lentivirus conferring puromycin resistance alone (Puro), control non-targeting shRNA (shCont) or shRNA targeting ADAM17 (A17-3 and A17-4). ADAM17 is the middle band, flanked by non-specific bands. B. Soluble FAS levels measured by ELISA in cell medium after 72 h from cells in A. Data are means ± SEM, n = 3. *** = P < 0.001, NS = Not Significant. C. Caspase-3 activity in hVSMC lysates from cell transduced with lentivirus expressing control shRNA or ADAM17 targeting shRNA (A17-3 and A17-4) infected with 300 pfu cell^-1 RAd60 or RAdT3-expressing adenovirus. Data are means ± SEM, n = 3. * = P < 0.05, ** = P < 0.01, NS = Not Significant. D. Quantification of surface FAS by single cell image analysis, data shows the mean number of spot-like FAS surface structures per cell, data are means ± SEM, n = 3. * = P < 0.05, *** = P < 0.001.</p

TIMP-3 increases FAS at the surface of hVSMCs at distinct regions.

<p>A. Soluble FAS (pg ml^-1) in the culture media of hVSMCs transduced with control (RAd60) or TIMP-3 (RAdT3)-expressing adenovirus at the time points indicated. Data are the mean ± SEM, n = 3. *** = P < 0.001. B. Surface levels of FAS analysed after 48 h by flowcytometry in hVSMCs infected with RAd60 or RAdT3-expressing adenovirus. Cells were stained with an IgM isotype control antibody or with anti-FAS IgM (mAb CH-11) followed by an anti-IgM Alexfluor-488 secondary antibody. Data is the mean fluorescence intensity ± SEM, n = 3, NS = Not Significant. C. Left; example hVSMCs in μ-slides stained with CellMask^TM Deep Red dye and for surface FAS with mAb CH-11 and an anti IgM Alexafluor-488 secondary antibody acquired by the iCys system. Middle; Image analysis contours superimposed on the left panel image generated by the iCys system. Contours in green are at the detection limit for the cell tracker dye, contours in blue are an enlargement of the contour in green to enable detection of the true cell periphery. Contours in white are the focal FAS surface staining detected. Right; Confocal image of surface staining of FAS with mAb CH-11 and an anti IgM Alexafluor-488 secondary antibody and counterstained with DAPI and Phalloidin-Alexafluor633. D–G. Quantification of FAS surface staining per cell in μ-slides by image analysis with the iCys system with approximately 70–100 cells detected per well. D. Analysis of total FAS fluorescence within the cell periphery analogous to the flowcytometry experiment in B. E. Number of spot-like FAS positive structures at the cell surface. F. Analysis of the intensity of staining within the spot-like FAS positive structures. G. Analysis of the area of spot-like FAS positive structures. Data are mean ± SEM, n = 6. * = P < 0.05, ** = P < 0.01, NS = Not Significant.</p

TIMP-3 induces the cellular redistribution of c-FLIP.

<p>Immunocytochemistry for c-FLIP and FAS in VSMCs infected with either control (RAd60) or RAdT3 adenovirus for 48 h. c-FLIP is shown in green, FAS in red and nuclei in blue (DAPI) in the overlay images. Scale bar represents 67 μm.</p

TIMP-3 induced apoptosis shows independence of FASL.

<p>A. hVSMCs were transduced with control adenovirus (RAd60) or adenovirus expressing TIMP-3 (RAdT3) for 48 h before staining with 2 μg ml^-1 isotype control IgG or an anti-FASL antibody and an anti-IgG Alexfluor488 conjugate. Cell-surface alexafluor488 mean fluorescence staining intensity was measured using flowcytometry. Data is the mean ± SEM, n = 3. NS = Not Significant. B. hVSMCs were transduced with control adenovirus (RAd60) or adenovirus expressing TIMP-3 (RAdT3) for 16 h before the addition of PBS (vehicle control), 2 μg ml^-1 isotype control IgG or a function blocking anti-FASL antibody. The antibody was added every 24 h before cell lysates were harvested 72 h post adenoviral transduction and caspase-3 activity measured. Data is the mean ± SEM, n = 3. *** = P < 0.001, NS = Not Significant. C. In order to demonstrate the function blocking FASL antibody could block FASL dependent apoptosis, hVSMCs were stimulated with either PBS vehicle control or 10 ng ml^-1 IFN-gamma for 24 h before the addition of FASL in membrane vesicles (vFASL), 2 μg ml^-1 isotype control IgG or a functional blocking FASL antibody. 24 h after the addition of antibody lysates were harvested and caspase-3 activity measured. Data are the mean ± SEM, n = 3. *** = P < 0.001, NS = Not Significant.</p

TIMP-3 induces a change in the cellular localisation of FAS and caspase-8 in hVSMCs.

<p>Immunocytochemistry for caspase-8 (red in overlay) and FAS (green in overlay) with nuclei in blue (DAPI) in the overlay images, in VSMCs infected with either control (RAd60) or RAdT3 adenovirus for 48 h. A and B are magnified images of RAd60-infected cells stained for caspase-8 and FAS shown in the upper panels. C and D are magnified images of RAdT3-infected cells stained for caspase-8 and FAS taken from the upper panels. Scale bars represent 36 μm in the upper panels and 7 μm in panels A to D.</p

TIMP-3 overexpression in hVSMCs induces apoptosis.

<p>A and B. Nuclear fragmentation measured by DAPI staining (examples in A for RAd60 and RAdT3, scale bar = 20 μm) expressed as percentage of total nuclei count for VSMCs infected with control adenoviral particles (RAd60), TIMP-3 expressing adenovirus (RAdT3) or Cys1-Ser TIMP-3 (RAd-(C1S)T3). Data are mean ± SEM, n = 3. *** = P < 0.001. C. Caspase-3 activity measured in hVSMC lysates at different time points post infection without or with RAd60, RAdT3 or RAd-C1S-T3. Data are mean ± SEM, n = 3. *** = P < 0.001. D. Western blot of hVSMC lysates harvested at the indicated time points probed for the 85 kDa caspase-3 cleavage product of PARP, TIMP-3 and re-probed for beta-actin as a loading control.</p

TIMP-3-induced apoptosis in hVSMCs is dependent on FAS, increases cellular FAS protein levels and induces DISC formation.

<p>A. Western blots of hVSMCs (Cont), transduced with lentivirus carrying puromycin resistance alone (Puro), expressing non-targeting control shRNA (shCont), or shRNA targeting FAS (FAS-1, -3, -4). B. Cells from (A) were infected with control (RAd60) or TIMP-3-expressing adenovirus (RAdT3) and caspase-3 activity analysed after 72 h. Data are the mean ± standard deviation, n = 3, *** = p < 0.001, NS = Not Significant. C. Western blots for FAS, FADD, FLIP, TIMP-3 and beta-actin in hVSMCs transduced with RAd60 or RAdT3 at the indicated time points. D. Western blots of VSMCs infected for 48 h with RAd60 or RAdT3 before immunoprecipitation of lysates with protein-G beads alone, IgG isotype control or anti-FAS monoclonal 3D5. Western blots were probed for caspase-8 then re-probed for FAS using anti-FAS IgM monoclonal CH-11. The band corresponding to procaspase-8 and the p18 form are indicated by Casp-8 and p18 Casp-8 respectively. A 26 kDa non-specific band was also detected. Empty lanes used to prevent carryover of the ECL signal on long exposure from RAd60 to RAdT3 immunoprecipitate containing lanes have been cropped from the image.</p

TIMP-3 overexpression induces apoptosis via a type-I death receptor pathway in hVSMCs.

<p>A. Western blot of lysates of VSMCs infected with either control (RAd60) or TIMP-3 (RAdT3)-expressing adenovirus harvested at the indicated times. Western blots were probed with anti-caspase-8, which can detect the pro, processed and 18 kDa active forms (Casp-8 and p18 Casp-8 respectively), anti-caspase-9 (Casp-9), anti-TIMP-3 and anti-beta-actin as a loading control. B and C. hVSMCs were infected with control (RAd60) or TIMP-3 (RAdT3)-expressing adenovirus followed by control (RAd60), Bcl2 (RAd-Bcl2), CrmA (RAd-CrmA) or dominant negative FADD (RAd-DN-FADD)-expressing adenovirus as described in the experimental procedures. Lysates were harvested after 72 h and analysed by Western blot to confirm expression of Bcl2, CrmA, DN-FADD and TIMP-3 with anti-beta-actin as a loading control (B) or caspase-3 activity measured (C). Data in C are the mean ± SEM, n = 5, *** = P < 0.001, NS = Not Significant. D and E. hVSMCs were infected with control (RAd60) or TIMP-3 (RAdT3)-expressing adenovirus for 48 h before incubation with both mitotracker green to measure total mitochondrial levels and TMRE to detect microchondrial depolarisation. RAd60 cells were also incubated with FCCP as a positive control for mictochondrial depolarisation. Mean fluorescence intensity ± SEM, n = 5 of mitotracker green (D) and TMRE (E) was measured using flowcytometry (example data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195116#pone.0195116.s001" target="_blank">S1B Fig</a>). * = P < 0.05, ** = P < 0.01, NS = Not Significant.</p