Inhibition of 4-HBA-mediated cell survival by suppressing HO-1.

<p>(A-B) C6 cells were transfected with HO-1 siRNA or control siRNA and 15 hrs later, were treated with 100 μM 4-HBA for 9 hrs. HO-1 levels were examined at 24 hrs after siRNA transfection (after 9 hrs of 4-HBA treatment) by immunoblotting. Representative photographs are presented (A) and the results obtained from three independent experiments are presented as means±SEMs (B). **p<0.01 versus siRNA-non-transfected/4-HBA treated cells. (C-D) C6 cells were transfected with HO-1 siRNA or control siRNA and 15 hrs after transfection treated with 100 μM 4-HBA for 9 hrs. Cells were then treated with serum-free media (C) or H₂O₂ (100 μM) (D) for 1 hr. Cell viabilities were determined by MTT assay 24 hrs after treating cells with serum-free media (C) or H₂O₂ (D) (48 hrs after HO-1 or control siRNA transfection). Changes in cell survivals observed in three independent experiments are presented as means±SEMs. **p<0.01 between indicated groups.</p

Induction of Nrf2-downstream genes by 4-HBA in C6 cells.

<p>(A-B) Cells were treated with 4-HBA (100 or 500 μM) for 3, 6, 9, or 12 hrs and protein levels of HO-1, NQO1, GCLM, and ɑ-tubulin were determined by immunoblotting. (C-D) Cells were pre-treated with 4-HBA (100 μM) for 6 or 9 hrs, treated with H₂O₂ (100 μM) for 1 hr, and protein levels of HO-1, NQO1, GCLM, and ɑ-tubulin were determined 1 hr later. (B, D) Protein levels determined in three independent experiments are presented as means±SEMs. **p<0.01, ^$$<0.01, ^#<0.05, ^## <i>p</i> < 0.01 between indicated groups.</p

Neuroprotective effects of GDNF and VEGF in 4-HCM.

<p>(A) C6 cells were treated with 250 μM of 4-HBA for 24 hrs and 4-HCM was collected and concentrated using a NANOSEP 10K filter. Primary neuronal cultures were treated with 200 μM of H₂O₂ for 30 min and LDH assays were carried out 24 hrs after H₂O₂ treatment. (B) Primary neuronal cultures were treated with 200 μM of H₂O₂ for 30 min and media were then replaced with 4-HCM. LDH assays were carried out 24 hrs later. (C-E) Primary neuronal cultures were pre-treated with 4-HCM for 4 hrs and then treated with 200 μM of H₂O₂ for 30 min. 4-HCM was pre-incubated with 1 μg/ml of anti-GDNF (C) or anti-VEGF (D) antibody or prepared from C6 cells transfected HO-1 siRNA (E). Non-specific IgGs and non-specific siRNA were used as negative controls. LDH assays were carried out 24 hrs after H₂O₂ treatment. Changes in cell death are presented as means±SEMs (n = 3). **p<0.01 versus the H₂O₂-treated control. VCM, vehicle-conditioned medium.</p

The up-regulation and nuclear translocation of Nrf2 by 4-HBA.

<p>(A, B) C6 cells were treated with 100 μM of 4-HBA for 3, 6, 9, or 12 hrs and total (A) and nuclear and cytoplasmic Nrf2 levels (B) were determined by immunoblotting. (C-D) Total (C) or nuclear (D) Nrf2 levels are presented as means±SEMs (n = 3). *p<0.05, **p<0.01 versus untreated controls, ^#P<0.05, ^## p< 0.01 between indicated groups. (E) Double fluorescent staining was performed using anti-Nrf2 antibody and DAPI. Nrf2-positive cells were identified using a rhodamine-conjugated secondary antibody. Arrows indicate Nrf2 translocation from cytoplasm to nucleus and arrowheads indicate the cytoplasmic localization of Nrf2. The photographs presented are representative of three independent experiments. The scale bar represents 100 μm.</p

Activations of ERK and Akt during the 4-HBA-mediated inductions of HO-1 and Nrf2 in C6 cells.

<p>(A-D) C6 cells were incubated with 100 μM 4-HBA for 3, 6, 9, or 12 hrs and levels of total or phosphorylated ERK and Akt were determined by immunoblotting with anti-ERK and anti-Akt or anti-pERK and anti-pAkt antibodies, respectively. (E-G) Cells were then preincubated with PD98059 (100 μM) or wortmannin (1 mM) for 60 min, treated with 100 μM 4-HBA for 9 hrs, and total HO-1 (E, G) and nuclear Nrf2 (F, G) levels were assessed by immunoblotting. Protein levels determined in three independent experiments are presented as means±SEMs. **p<0.01 versus treatment naïve control cells, ^#<i>p</i> <0.05, ^## <i>p</i> < 0.01 between indicated groups.</p

Accumulations of GDNF and VEGF in 4-HBA-conditioned astrocyte culture media.

<p>(A-B) C6 cells were incubated with 50, 100, or 250 μM of 4-HBA for 24 hrs and GDNF or VEGF protein levels in media were assayed using commercial kits. (C-D) C6 cells were transfected with HO-1 siRNA or control siRNA, and 12 hrs later, treated with 250 μM 4-HBA for 24 hrs. Secreted GDNF and VEGF protein levels in media were then measured. *p<0.05, versus the untreated control (n = 3), ^##p<0.01 versus the 4-HBA-treated control (n = 3).</p

Autocrine protective effects of GDNF and VEGF in 4-HCM.

<p>(A) 4-HCM was collected as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177322#pone.0177322.g007" target="_blank">Fig 7A</a>. (B) C6 cells were pre-treated with 4-HCM for 4 hrs and then treated with 100 μM of H₂O₂ for 60 min. 4-HCM was pre-incubated with 1 μg/ml of anti-GDNF or anti-VEGF antibody or prepared from C6 cells transfected with HO-1 siRNA before being administered to C6 cells. Non-specific IgG was used as a negative control. MTT assays were carried out 24 hrs after H₂O₂ treatment. Changes in cell viability are presented as means±SEMs (n = 3). **p<0.01 versus 4-HCM-pretreated/H₂O₂-treated cells. VCM, vehicle-conditioned media.</p

Protection of H₂O₂-treated C6 cells by 4-HBA.

<p>(A-B) C6 cells were treated with H₂O₂ (100 μM) for 1 hr in the presence or absence of 4-HBA (25, 50, 100, or 250 μM) (A), or pre-treated with 4-HBA (25, 50, 100, or 250 μM) for 3 hrs and then treated with H₂O₂ (100 μM) for 1 hr (B). (C) C6 cells were pre-treated with 4-HBA (100 μM) for 3, 6, or 9 hrs and then treated with H₂O₂ (100 μM) for 1 hr. In all experiments, MTT assays were carried out 24 hrs after H₂O₂ treatment. Changes in cell survival are presented as means±SEMs (n = 3). **p<0.01 versus untreated controls, ^#p<0.05 and ^##p<0.01 between indicated groups.</p