Additional file 1: Figure S1. of Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta

Dot blot analyses using species-specific antibodies. Preparation of Aβ contains both oligomeric and fibrillar Aβ. Immunoreactivity of dot blots of Aβ scrambled (Aβscr) or Aβ1-42 was detected using 4G8, A11, or OC antibodies as described in the “Methods” section. Scrambled Aβ demonstrates antibody specificity. (PDF 155 KB

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

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

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

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 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

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

Inhibitory effect of nicardipine on LPS/IFN-γ- or peptidoglycan-stimulated iNOS and COX-2 expressions.

<p>(A and B) BV-2 microglial cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 24 h. (C and D) Cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of peptidoglycan (10 μg/ml) for another 24 h. Western blot analysis for iNOS (A and C) and COX-2 (B and D) expression was performed on whole cell lysates. The quantitative results are shown in the bottom panels. iNOS expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated-group and the group treated LPS/IFN-γ (or peptidoglycan) with nicardipine (one-way ANOVA followed by Bonferroni's post hoc test). COX-2 expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated- group and the LPS/IFN-γ (or peptidoglycan) with nicardipine treated- group (one-way ANOVA followed by Bonferroni's <i>post hoc</i> test). The results are expressed as mean ± S.E.M. from 4 to 5 independent experiments. *, <i>p</i><0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.</p

Nicardipine prevents LPS-induced microglial activation.

<p><b><i>Mice received</i></b> intraperitoneal injections of nicardipine at concentrations of either 5/kg or 50 mg/kg, <b><i>once per day</i></b>, for <b><i>3 consecutive days. On the third day</i></b>, nicardipine treatment was followed with a single <b><i>intraperitoneal injection</i></b> of LPS (20 mg/kg). Microglial morphology was visualized by anti-Iba-1 immunolabeling and DAB (n = 5 each group).</p