Involvement of PKC-δ and p38 signaling pathways in crotonaldehyde-stimulated HO-1 expression.

<p>Cells were pretreated with PD 98059 (MEK1 inhibitor), LY 294002 (Akt/PI3K inhibitor), SP 600125 (c-Jun N-terminal Kinases (JNKs) inhibitor), SB 203580 (p38 inhibitor) or Rottlerin (PKC-δ inhibitor) for 1 h, followed by incubation with 25 µM crotonaldehyde for 16 h. Whole cell lysates were prepared and subjected to western blot analysis with antibodies against anti-HO-1 and GAPDH, as indicated (A and C). HepG2 cells were pretreated with inhibitors at the indicated concentrations for 1 h; followed by incubation with 25 µM crotonaldehyde for 2 h. Total RNA was prepared and subjected to RT-PCR for HO-1 and GAPDH (B and D). Cell lysates were immunoblotted with antibodies for the phosphorylated form of p38 MAPK and PKC-δ (E and F). Transient transfection of cells with PKC-δ or p38 siRNA inhibited up-regulation of crotonaldehyde-stimulated HO-1 protein expression (G and H). Representative Western blots of three independent experiments are shown. +, crotonaldehyde alone treated group.</p

Effect on cell cycle distribution after treatment with ZnPP or HO-1 siRNA in crotonaldehyde-stimulated HepG2 cells.

<p>Cell cycle analysis was performed by PI staining. HepG2 cells were treated with 25 µM crotonaldehyde and absence or presence of 1 µM ZnPP or HO-1 siRNA for 16 h. After treatment, the cells were stained with propidium iodide. Fluorescence activated cell sorting (FACS) analysis using PI staining was performed for DNA content measurement. Apoptosis was measured as the percentage of total cell population with the sub-G1 DNA content and was in the region labeled M1. Results are expressed as a dot plot and represent three independent experiments.</p

Effect of crotonaldehyde-induced HO-1 inhibition on cell death.

<p>Cells were incubated in the absence or presence of ZnPP or HO-1 siRNA for 16 h before the indicated tests were performed. Crotonaldehyde-stimulated HepG2 cells were pretreated for 1 h with 1 µM ZnPP or HO-1 siRNA. Protective effect of HO-1 induction on cell death as determined by <i>in situ</i> terminal nick end-labeling (TUNEL). Treatment with H₂O₂ (0.5 mM) served as a positive control. Representative images illustrating fluorescent TUNEL (green) staining of cells cultured for 16 h before the indicated tests were performed (A). The graph indicates that inhibition of HO-1 expression in crotonaldehyde-stimulated HepG2 cells show a significant increase in the number of TUNEL-positive cells compared with those of normal and crotonaldehyde-treated cells (B). TUNEL-positive cells were quantified in five random fields in each culture well, and converted in percentages by referring to the total number of cells. Data represent the mean ± SD of three independent experiments. *<i>p</i><0.005 vs. CRA 25 µM treated cells.</p

Crotonaldehyde-stimulated induction of HO-1 expression is mediated by Nrf2-EpRE/ARE.

<p>Cells were treated with crotonaldehyde at the indicated concentrations for 4 h. Nuclear extracts were prepared, and protein samples (40 µg) were subjected to Western blotting using an anti-Nrf2 antibody or an anti-Lamin B (a nuclear protein marker) antibody (A). Transient transfection of HepG2 cells with Nrf2 specific siRNA inhibited expression of the HO-1 protein. Nrf2 siRNA abrogated induction of crotonaldehyde-stimulated HO-1 protein expression (B). +, crotonaldehyde alone treated group. Cells transfected with an EpRE/ARE-luciferase construct were treated with various concentrations of crotonaldehyde for 4 h, and the lysates were mixed with a luciferase substrate. A luminometer was used to measure luciferase activity (C). Data represent the mean ± SD of 4 independent experiments. *<i>p</i><0.001 vs. control.</p

Effect of crotonaldehyde (CRA) on HO-1 expression.

<p>Cells were incubated with the indicated concentration of crotonaldehyde for 16 h. Protein in cell lysates was analyzed by Western blot using HO-1 specific antibody (A). RT-PCR was conducted to measure the levels of HO-1 mRNA transcript (C). Cells were harvested at various time intervals (B and D). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were measured to ensure equal amounts of protein and mRNA loaded. Cell viability was estimated by the MTT method (E). Data represent the mean ± SD of results in three independent experiments.</p