DSA imaging of treated tumors.

<p>The tumors showed hypervascularity in the liver as determined with DSA imaging (A, black arrow). After lipiodol (60°C) injection, the tumors are completely or largely de-vascularized and show on DSA as a lipiodol-filling defect (B, black arrow).</p

Expression of PCNA protein.

<p>As detected through immunohistochemistry, PCNA protein expression was detected mainly in viable VX2 tumor cells (brown; A, control 400<b>×</b>; B, treated 400<b>×</b>).</p

Expression of VEGFR and VEGF protein and mRNA levels.

<p>The relative changes in VEGFR or VEGF protein and mRNA levels in tumor tissue were detected after treatment in each group (n = 10). A and B. VEGFR and VEGF mRNA expression levels were evaluated using real-time quantitative PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061583#s2" target="_blank">Materials and Methods</a> section. C and D. VEGFR and VEGF protein levels were detected using Western blot analysis (upper panel). β-actin was detected as a loading control. VEGFR and VEGF expression levels were quantified through densitometry and plotted as the fold change (lower panel). The values are presented as the mean±SD of 3 independent experiments (* <i>P</i><0.05 <i>vs.</i> control group).</p

TEM results of treated tumors.

<p>Stimulated by heated (60°C) lipiodol perfusion, TEM results revealed that the tumor endothelial cell microvilli decreased (A, 5000×), the vascular endothelial cell endoplasmic reticulum expanded, and the chondriosome was swollen (B, 10000×).</p

Comparisons of PCNA protein levels between groups after perfusion.

<p>PCNA: control <i>vs.</i> treated group, <i>P</i> = 0.047.</p

Effect of PQQ on Microglia Activation in the Brain.

<p>Representative photographs show Iba-1 immuno-stained microglia of the mouse brain (A). LPS group shows mostly an activated-form of microglia which display an increased size, irregular shape, thickened and shortened processes and intensified Iba-1 immunostaining density, in the dentate gyrus of hippocampus (DG) and cerebral cortex (Cortex) compared to the normal group. Moreover, both PQQ treated groups show a decrease of morphological activation of microglia in all brain regions with respect to LPS group. Representative DG (a–d) and Cortex (e–h) immunostained slices are presented at ×40. High power images of Iba-1-expressed microglia in the boxed regions in (f), (g) and (h) are also shown at×200 (i–l). Arrow indicates the Iba-1-expressed microglia. The number of microglia was counted and normalized in the corresponding same area (B). LPS increases the number of Iba-1-expressed microglia in the brain. PQQ treatment significantly reduces the number of Iba-1-expressed microglia both in the cerebral cortex and DG. The results shown are mean ± S.E.M. (<i>n</i> = 6 in each group) of three independent experiments. <i>*p<0.05 vs. control group, #p<0.05 vs. only LPS group.</i></p

Effects of PQQ on LPS-induced NO, PGE2 production and iNOS, COX-2 expression in microglia cells.

<p>Primary microglia were pretreated with or without PQQ for 1 h followed by LPS (100 ng/ml) treatment for indicated durations. Culture supernatants were collected 24 h later, the released NO in primary microglia (A) was determined by the Griess assay, total mRNA was harvested 6 h later, and the mRNA level of iNOS (B) and COX-2 (D) was measured by real-time PCR. (C) Concentrations of PGE2 in the culture supernatants of primary microglia were determined by ELISA. (E) 24 h later, the protein level of iNOS and COX-2 were detected by western blot analysis in primary microglia cells. The results presented as mean ± S.E.M. of at least three independent experiments, <i>*p<0.05 vs. control group, #p<0.05 vs. only LPS group.</i></p

Effects of PQQ on LPS-induced MAP kinases activity in microglial cells.

<p>(A) Microglial cells were treated with or without PQQ 1 h prior to 4 h co-treatment with LPS. The protein expression of p-p38 and p-JNK was determined by western blot analysis. Non-phosphorylated form of each targeted protein was used as loading control. (B) Bar graph represents the average levels of p-p38 and p-JNK compared to the control. The results shown are mean ± S.E.M. of three independent experiments. <i>*p<0.05 vs. control group, #p<0.05 vs. only LPS group.</i></p

Effects of PQQ on LPS-stimulated expression of pro-inflammatory mediators in microglial cells.

<p>Cells were treated with the indicated concentrations of PQQ 1 h prior to 6 h co-treatment of LPS (100 ng/ml). Primary microglia were harvested and total RNA was prepared. The mRNA expression of pro-inflammatory mediators: TNF-a, IL-1β, IL-6, COX-2, MCP-1 and MIP-1α was measured by real-time PCR. GAPDH was used as an internal control. The results shown are mean ± S.E.M. of three independent experiments. <i>*p<0.05 vs. control group, #p<0.05 vs. only LPS group.</i></p

Effects of PQQ on LPS-induced p65 and NF-κB activity in microglial cells.

<p>(A) Representative images of NF-κB p65 in microglial cells of each group. Cells were pretreated with or without PQQ for 1 h followed by 100 ng/ml LPS treatment for 2 h. Microglial cells were incubated with NF-κB p65 antibody and immunofluorescence microscopy was used to visualize the localization of NF-κB p65 (Green; a-c), boxed regions in (a-c) are also shown at×200 (j-m). Nuclei were visualized using DAPI counterstaining (Blue; d-f). (B) Cells were treated with 100 ng/m LPS for indicated time. p65 protein level was measured by western blot analysis. Non-phosphorylated p65 was used as loading control, and the expression of p-p65 was normalized to control and quantified by densitometric analysis. The results shown are mean ± S.E.M. of three independent experiments. <i>*p<0.05 vs. control group, #p<0.05 vs. only LPS group.</i></p