Activation of caspase-3 and PARP cleavage.

<p>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.</p

A) <i>HA-D4 transfection</i>.

<p>pcDNA3-HA-D4 and the control plasmid pcDNA3-HA were transfected in both Ins-1E_PED/PEA-15 and in Ins-1E_CTRL cells, and D4 expression was analyzed by qualitative PCR. <b>B) </b><b><i>Apoptosis.</i></b> 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. ***<i>p</i><0.001.</p

Apoptosis in isolated mice islets.

<p>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. ***<i>p</i><0.001.</p

PARP cleavage.

<p><b>A</b>) 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>B</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. *<i>p</i><0.05, **<i>p</i><0.01.</p

TUNEL analysis.

<p>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. *<i>p</i><0.05 and ***<i>p</i><0.001.</p

The effects of <i>CA</i>de on gene expression during adipogenesis in 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated into mature adipocytes for 8 days, as described under “Materials and Methods”, in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml). At the indicated time points, cells were collected for the extraction of RNA. qPCR was performed to detect the mRNA expression of (<b>A</b>) <i>C/ebpβ</i> at D2, (<b>B</b>) <i>Pparγ</i> at D4, and (<b>C</b>) <i>Glut4</i> and (<b>D</b>) <i>Fapb4</i> at D8 of the adipogenesis. Results are means ± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using Student’s t-test. **<i>p</i><0.01, and ***<i>p</i><0.001 <i>vs</i>. Ctrl at D2, D4 or D8.</p

PLD-1 role in PED/PEA-15 anti-apoptotic action.

<p>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-1E_CTRL cells). Each point is the mean value from eight identical wells. Values represent the mean ±SD of three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001. <b>C</b>) 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. *<i>p</i><0.05, ***<i>p</i><0.001.</p

Apoptosis-Related Genes Expression.

<p>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. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001.</p

The effects of <i>CADE</i> on cell cycle progression during adipogenesis in 3T3-L1.

<p>MCE was induced in 3T3-L1 pre-adipocytes with differentiation medium, as described under “Materials and Methods”. Cells were then harvested at 12, 14, and 16 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml) and stained with PI solution for flow cytometer cell cycle analysis. <b>A</b>) Histograms of cell cycle distribution in G₀/G₁, S or G₂/M phases. <b>B</b>) Quantitative analysis of cell cycle distribution. The results are means ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test *<i>p</i><0.05, and ***<i>p</i><0.001, <i>CA</i>de S Phase <i>vs</i>. Ctrl S Phase; ^#<i>p</i><0.05, <i>CA</i>de G₂/M Phase <i>vs</i>. Ctrl G₂/M Phase.</p

The effects of <i>CADE</i> on <i>C/ebpβ</i> gene expression and CREB activation during the early stage of adipogenesis in 3T3-L1.

<p>Adipogenesis was induced in 3T3-L1 pre-adipocytes with the differentiation medium (MDI), as described under “Materials and Methods”. Cells were then harvested at 1, 2, 4 and 8 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml) and processed for qPCR and western blot analysis. <b>A</b>) qPCR of <i>C/ebpβ</i> mRNA expression. Results are means ± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using one-way ANOVA. ***<i>p</i><0.01, <i>vs</i>. 3T3-L1 cells at 0 h; ^###<i>p</i><0.001, <i>CA</i>de 2 h <i>vs</i>. Ctrl 2 h; ^<i>¶</i><i>p</i><0.05, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h; ^†††<i>p</i><0.001, <i>CA</i>de 8 h <i>vs</i>. Ctrl 8 h. <b>B</b>) The representative western blot show levels of the total and Ser¹³³ phosphorylated form of the cAMP response element-binding protein (CREB) and of the β-Actin protein. <b>C)</b> CREB protein binding on <i>C/ebpβ</i> promoter was evaluated by ChIP analysis on 3T3-L1 cells harvested at 4 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml). ChIP enrichment is relative to input chromatin. Data are expressed as mean ± SD of values from at least three independent experiments. Statistical analysis was performed using Student’s t-test. **<i>p</i><0.01, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h.</p