Figure 1.
PPARα ligands attenuate H2O2-induced VSMC apoptosis.
(A–C) A-10 VSMCs were pretreated with Wy14,643 (50 µM or otherwise indicated), or cPGI2 (100 µM) followed by H2O2 (0.8 mM). (A) Cells were stained with DAPI or immune-stained for cleaved caspase 3 and examined under fluorescent microscope. Scale bar = 100 µm. (B and C) Cleaved caspase 3 was analyzed by Western blotting. Upper panel shows a representative blot and the lower panel shows the quantitative analysis of densitometry of the Western Blots. (D) A-10 cells were pretreated with MK886 (25 µM) or GSK3787 (25 µM) followed by cPGI2 (100 µM) and H2O2 (0.8 mM). Upper panel shows a representative blot and the lower panel shows the quantitative analysis. (E) A-10 cells were transfected with Flag-tagged PPARα vectors at different concentrations. PPARα expression was analyzed using a Flag antibody. Cleaved caspase 3 was analyzed by Western blotting using a specific antibody. The left panel shows a representative Western blot and the right panel shows the quantitative analysis. Each error bar denotes mean±SEM and all blots are representative of n≥3. NS denotes statistically non-significant.
Figure 2.
PPARα rescues H2O2-induced depression of 14-3-3β and θ levels.
(A) A-10 cells were pretreated with Wy14,643 (50 µM) followed by H2O2 (0.8 mM). 14-3-3 proteins were analyzed by Western blotting. Left panel shows representative Western blots and right panel shows densitometry analysis. (B) Cells were pretreated with caspase 3 inhibitor, Z-DEVD-fmk (20 µM) followed by H2O2. 14-3-3β and θ were analyzed by Western blotting. (C) Cells were treated with Wy14,643 (50 µM) or GW9578 (5 µM) and changes in 14-3-3 proteins were analyzed. (D) Cells were transfected with PPARα vectors and 14-3-3 proteins were analyzed. Each error bar denotes mean±SEM and all blots are representative of n≥3.
Figure 3.
PGI2 increases 14-3-3β expression.
(A) A-10 cells were transfected with Ad-COPI or control adenoviral vector Ad-null. 14-3-3 proteins were determined by Western blotting. Left panel shows representative Western blots and right panel shows densitometry analysis. (B) Cells were treated with cPGI2. 14-3-3β was measured by Western blotting. (C) Ad-COPI or Ad-null transfected cells were treated with H2O2. 14-3-3 isoforms and cleaved caspase 3 were analyzed. Left panel shows representative Western blots and right panel shows densitometry analysis. Each error bar denotes mean±SEM and all blots are representative of n≥3.
Figure 4.
14-3-3β protects against H2O2-induced apoptosis by sequestering Bad.
(A) A-10 cells were transfected with Flag-tagged 14-3-3β, ε or θ vectors. Following H2O2 treatment, cells were lysed and cleaved caspase 3 was determined. (B) Cells were transfected with 14-3-3β siRNA or control scRNA. The transfected cells were treated with cPGI2 and H2O2. 14-3-3β and cleaved caspase 3 were analyzed by Western blotting. (C) Cells were treated with cPGI2 (100 µM) or Wy14,643 (50 µM) prior to H2O2 treatment. Mitochondrial fractions of VSMCs were isolated and Bad was analyzed by Western blotting. Heat shock protein 60 (HSP60) was concurrently measured as mitochondrial marker. (D) Cells were treated with cPGI2 (100 µM) or Wy14,643 (50 µM) and then lysed. Lysates were immunoprecipitated with Bad antibody or control IgG. 14-3-3β and Bad in the immunoprecipitates were determined by Western blotting. Each error bar denotes mean±SEM and all blots are representative of n≥3. NS denotes statistically non-significant.
Figure 5.
H2O2 degrades VSMC contractile proteins and SRF via caspase 3.
(A) Cells were pretreated with Z-DEVD-fmk (20 µM) followed by H2O2. SRF and contractile proteins were analyzed by Western blotting. (B) Cells were pretreated with cPGI2 (100 µM) followed by H2O2. SRF and contractile proteins were analyzed by Western blotting. (C) Immunofluorescent staining of SM22α and nuclear staining with DAPI in H2O2-treated cells in the absence and the presence of cPGI2 or Wy14,643. Scale bar = 100 µm. (D) VSMCs were transfected with Flag-tagged 14-3-3β or θ vectors. Following H2O2 treatment, cells were lysed and SM22α and calponin-1 were determined by Western blotting. Each error bar denotes mean±SEM and all blots are representative of n≥3.
Figure 6.
Prostacyclin prevents contractile protein reduction induced by combined growth factors (GFs).
(A) Cells were pretreated with cPGI2 followed by GFs. (B) VSMCs were transfected with 14-3-3β, η or θ siRNA or a control scRNA and the respective 14-3-3 proteins were analyzed. (C) VSMCs transfected with siRNA of 14-3-3β, η or θ were treated with cPGI2 (100 µM) and GFs. SM22α and Calponin-1 in cell lysates were analyzed by Western blotting. (D) VSMCs were transfected with Flag-tagged 14-3-3β or θ vectors. Following GFs treatment, cells were lysed and SM22α was determined by Western blotting. All blots are representative of n≥3. (E) A schematic illustration of the role of PGI2/PPARα/14-3-3β and θ in controlling VSMC apoptosis and contractile phenotype.