Role of HO-1 in neuroprotective effect in Mes23.5 dopaminergic neurons.

<p>Cells were treated with Copp IX (1 µM) (A and C) or hemin (B and D) for 8 h followed by treatment with 6-OHDA (50 µM) for another 16 h. Cell viability was determined by MTT assay and SRB assay. Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with vehicle group. #, <i>p</i><0.05 as compared with desipramine-treated group.</p

Desipramine induces Nrf2 translocation from cytoplasm to nucleus in Mes23.5 dopaminergic neurons.

<p>Cells were incubated with desipramine (20 µM) for indicated time periods (4 or 8 h), and Nrf2 expression levels in whole cell lysates (A) and nuclear extracts (B) were determined by immunoblotting with Nrf2-specific antibody. PCNA was used as the loading control for nuclear fraction. The quantitative data are shown in below. The Nrf2 expression is significantly different between desipramine treatment group and control group in nuclear extract (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. Cells were treated with or without desipramine (20 µM) for 2 h, and the levels of Nrf2 were determined by immunoflourescence (C). Note that the Nrf2 translocates from cytoplasm to nucleus in response to desipramine stimulation. Results are the representatives of three independent experiments.</p

Desipramine and fluoxetine increase HO-1 expression in Mes23.5 dopaminergic neurons.

<p>Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (A) or fluoxetine (C) for 24 h or with desipramine (20 µM; B) or fluoxetine (20 µM; D) for the indicated time periods (4, 8 and 24 h). Whole cell lysates were extracted and subjected to Western blot for detection of HO-1 expression. The HO-1 expression is significantly different between control group and desipramine or fluoxetine treatment groups (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (E) or fluoxetine (G) for 8 h or with desipramine (20 µM; F) or fluoxetine (20 µM; H) for indicated time periods (4, 8 and 24 h). The quantitative data are shown in below. HO-1 mRNA expression was determined by RT-PCR. Results are the representatives of three independent experiments.</p

Desipramine increases HO-1 expression through Nrf2 activation in Mes23.5 dopaminergic neurons.

<p>(A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.</p

Neuroprotection of desipramine on rotenone- and 6-OHDA-induced neurotoxicity.

<p>Mes23.5 dopaminergic neurons were pretreated with various concentrations of desipramine for 8 h and followed by treatment with rotenone (3 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (A and C, respectively). The neuroprotective effects are significantly different between rotenone alone group and rotenone treated with desipramine group in both MTT and SRB assays (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle group. #, <i>p</i><0.05 as compared with the desipramine-treated group. Cells were pretreated with various concentrations of desipramine for 8 h and followed by treatment with 6-OHDA (50 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (B and D, respectively). The neuroprotective effect is significantly different between 6-OHDA alone group and 6-OHDA treated with desipramine group in SRB assay (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle group. #, <i>p</i><0.05 as compared with the desipramine-treated group. Whole cell lysates were subjected to Western blot for detection of tyrosine hydroxylase (TH). Results are the representatives of at least three independent experiments.</p

Involvement of HO-1 in desipramine-mediated neuroprotective effect in Mes23.5 dopaminergic neurons.

<p>Cells were treated with ZnPP IX (0.1 or 0.3 µM) for 30 min and follow by treatment with desipramine for 8 h, and then treated with rotenone (A and C) or 6-OHDA (B and D) for another 16 h. Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with vehicle group.</p

ERK and JNK signaling pathways are involved in desipramine- and fluoxetine-increased HO-1 expression in Mes23.5 dopaminergic neurons.

<p>Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.</p

PKCα expression and cell proliferation/migration/invasion inhibition by the truncated-MZF-1 (MZF-1ΔDBD) or-Elk-1 (Elk-1ΔDBD) proteins in SK-Hep-1 cells.

<p>The inhibitory effects of transfection with MZF-1ΔDBD and Elk-1ΔDBD constructs from RT-PCR (A) and Western blot analysis (B) assays. SK-Hep-1 cells were transiently transfected with FLAG vector (5 μg), c-Myc vector (5 μg), FLAG-MZF-1ΔDBD (5 μg), or c-Myc-Elk-1ΔDBD (5 μg). (C and D) Effect of MZF-1ΔDBD and Elk-1ΔDBD on cell growth. The SK-Hep-1 cell was transfected with the indicated dose of MZF-1ΔDBD and Elk-1ΔDBD for 1 and 2 d. Untreated cultures were designated as control (Control). Absorbance values obtained from untreated cells on day 0 after subculture were considered 100%. The migration (E and F) and invasion (G and H) assays were performed on cell cultures treated with 5 μg dose of FLAG vector, MZF-1ΔDBD, c-Myc vector, and Elk-1ΔDBD, as described in the Materials and Methods Section. Values are presented as means ± SE of three replicates from two independent experiments. **, P < 0.01 vs. FLAG or c-Myc vector-transfected group.</p

MZF-1 and Elk-1 form a complex at the MZF-1 and Elk-1 sites of the PKCα promoter.

<p>(A) Interaction between endogenous MZF-1 and Elk-1 proteins in SK-Hep-1 cells. Protein extracts underwent immunoprecipitation (IP) with anti-MZF-1, anti-Elk-1, or control rabbit IgG, as indicated. Proteins were resolved via SDS-PAGE and then underwent immunoblotting (IB) using anti-MZF-1 or anti-Elk-1 antibodies. (B) N-terminal domain of MZF-1 interacts with Elk-1 at the C-terminal domain of the protein. SK-Hep-1 cells were transfected with expression vectors for FLAG-MZF-1ΔDBD (5 μg) or c-Myc-Elk-1ΔDBD (5 μg), as indicated. Protein extracts underwent IP with anti-Myc and anti-FLAG, as indicated. Proteins were resolved via SDS-PAGE and underwent IB using an anti-MZF-1 or anti-Elk-1 antibody. (C) ChIP and Re-ChIP assays. In the ChIP assay (left), the chromatin was pulled down with IgG, MZF-1, and Elk-1 antibodies, whereas in the Re-ChIP assay (right), the pulled down chromatin interacted with Elk-1 and then with MZF-1 antibodies, after which the sequence was reversed. The PCR products shown are the 210 bp bands in the PAGE result. The input represents the purified chromatin for parallel PCR reaction as a positive control. (D) ChIP assay performed on SK-Hep-1 cells transfected with 5 μM sense or antisense of MZF-1 or with 5 μM sense or antisense of Elk-1. PCR was performed on purified chromatin as control (Input) and on chromatin fragments from the IP with the specific antibodies. The data represent 1 of 3 independent experiments with similar results.</p

Activation of PKCα expression by combinations of MZF-1 and Elk-1 directly binding to the promoter.

<p>(A) Transcriptional activation of PKCα promoter activity by wild-type MZF-1 and Elk-1 and DNA-binding deficient mutants MZF-1 (MZF-1ΔDBD) and Elk-1 (Elk-1ΔDBD) via luciferase assays. MZF-1 and Elk-1 synergistically enhance PKCα transcriptional activity when co-transfected to Huh-7 and HepG2 cells, whereas co-transfection of the DNA-binding domain deletion mutant of MZF-1 (MZF-1ΔDBD)/Elk-1 (Elk-1ΔDBD) failed to enhance the PKCα transcriptional activity. Luciferase activity was normalized to the level of β-galactosidase. Transcriptional activity is expressed as fold induction compared with the level obtained with each reporter vector in the presence of empty pcDNA 3.1/<i>Myc</i>-His vectors. The results are the mean ± SE of three independent experiments performed in triplicate. (B) MZF-1 and the Elk-1 binding sites regulate the PKCα promoter activity in Huh-7 cells. Cells were co-transfected with different mutant PKCα promoter luciferase constructs and vectors containing MZF-1 and Elk-1 transcription factors. Luciferase activity was normalized to the level of β-galactosidase. The results are the mean ± SE of three independent experiments performed in triplicate. **, P < 0.01 versus the cells transfected with normal PKCα promoter constructs in each group.</p