Figure 1.
Apoptosis in isolated mice islets.
Mouse pancreatic islets were treated or not with 10 µM hydrogen peroxide for 16 hours. After this, histone-associated DNA fragments were quantified by ELISA to evaluate apoptotic cell death. Each column represents the mean ± SE from three separate experiments. ***p<0.001.
Figure 2.
A) Representative microscopic views of apoptosis after the TUNEL staining (image scale 10X). Cells were treated for 48 hours with 70 µM hydrogen peroxide and then stained with TUNEL reagent and DAPI to detect apoptotic (green) and total nuclei (blue), respectively. B) Bar graph represents the quantification of apoptosis by TUNEL assay. Results are quantified as the percentage of TUNEL-positive cells in 4 high magnification fields per slide. Values represent the mean ±SD of three independent experiments. *p<0.05 and ***p<0.001.
Figure 3.
Activation of caspase-3 and PARP cleavage.
A) Caspase-3 activation. Cells were grown on a glass coverslips and treated or not with 70 µM hydrogen peroxide for 48 hours. After fixation and permeabilization, cells were processed for anticaspase-3 antibody (in red). Arrows indicate cells stained with antibody to the active form of caspase-3. The total number of cells is visualized by the background staining. The results shown are representative of 3 independent experiments. B) PARP cleavage. Cells were treated or not for 48 hours with 70 µM hydrogen peroxide, as indicated. Whole-cell extracts (30 µg) were analyzed by Western blot with anti-PARP antibody. Full-length PARP (113 kDa) and the cleaved fragment (89 kDa) are indicated by arrowheads. Tubulin was used as loading control (n = 3). A representative image is shown.
Figure 4.
Apoptosis-Related Genes Expression.
Cells were treated or not with 70 µM hydrogen peroxide for 48 hours. Total RNA was then isolated and the mRNA levels of Bcl-xL (A), Bcl-xS and Bad (B) genes were assessed by real-time RT-PCR using 18S as internal control. Values represent the mean ±SD of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.
Figure 5.
PLD-1 role in PED/PEA-15 anti-apoptotic action.
A) PKC alpha phosphorylation. Cells were treated or not for 48 hours with 150 µM propranolol, as indicated. Whole-cell extracts (30 µg) were analyzed by Western blot with anti-phospho PKC alpha antibody. Tubulin was used as loading control (n = 3). A representative image is shown. B) Cell viability. Cells were cultured in 96-well cell culture plates, pretreated with 150 µM propranolol and then incubated with 70 µM hydrogen peroxide as indicated. After 48 hours, cell viability was estimated by use of the sulforhodamine B assay. Cell survival is expressed as the percent of control (untreated Ins-1ECTRL cells). Each point is the mean value from eight identical wells. Values represent the mean ±SD of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001. C) Apoptosis. Cells were pretreated with 150 µM propranolol and then incubated with 70 µM hydrogen peroxide as indicated. After 48 hours, cells were harvested and apoptosis was quantitated by evaluating the level of DNA fragmentation using the Roche Cell Death Detection ELISAPLUS. Data are the mean value of four identical wells. Values represent the mean ±SD of three independent experiments. *p<0.05, ***p<0.001.
Figure 6.
A) Representative microscopic views of apoptosis after the TUNEL staining (image scale 10X). Cells were pretreated with 150 µM propranolol and then treated for 48 hours with 70 µM hydrogen peroxide and then stained with TUNEL reagent and DAPI to detect apoptotic (green) and total nuclei (blue), respectively. B) Bar graph represents the quantification of apoptosis by TUNEL assay. Results are quantified as the percentage of TUNEL-positive cells in 4 high magnification fields per slide. Values represent the mean ±SD of three independent experiments. **p<0.01 and ***p<0.001.
Figure 7.
A) Cells were pretreated with 150 µM propranolol and then treated or not for 48 hours with 70 µM hydrogen peroxide as indicated. Whole-cell extracts (30 µg) were analyzed by Western blot with anti-PARP antibody. Full-length PARP (113 kDa) and the cleaved fragment (89 kDa) are indicated by arrowheads. Tubulin was used as loading control (n = 3). A representative experiment is shown. B) The bands for cleaved PARP were scanned, densitometrically quantitated using NIH Image J software and the resulting data were plotted on the bar graph. Values represent the mean ±SD of three independent experiments. *p<0.05, **p<0.01.
Figure 8.
pcDNA3-HA-D4 and the control plasmid pcDNA3-HA were transfected in both Ins-1EPED/PEA-15 and in Ins-1ECTRL cells, and D4 expression was analyzed by qualitative PCR. B) Apoptosis. Cells were incubated with 70 µM hydrogen peroxide as indicated. After 48 hours, cells were harvested and apoptosis was quantitated by evaluating the level of DNA fragmentation using the Roche Cell Death Detection ELISAPLUS. Data are the mean value of four identical wells. Values represent the mean ±SD of three independent experiments. ***p<0.001.