SU5416 directly inhibits nNOS enzyme activity in a concentration-dependent manner.

<p>The inhibitory effects of SU5416 on rat cerebellum nNOS were shown in the graph. The IC₅₀ value was also indicated. Each individual point was an average from three independent experiments.</p

SU5416 attenuates the deficit of locomotion behavior on zebrafish larval induced by MPTP.

<p>One dpf zebrafish embryos were treated with 200 µM MPTP for 2 days, and then co-incubated with 10 µM MPTP and SU5416 or VRI at the indicated concentrations for 72 hours, and zebrafish larval co-treated with MPTP and 150 µM L-dopa or 20 µM L-deprenyl were used as positive controls. After treatment, zebrafish were collected to perform locomotion behavior test using Viewpoint Zebrabox system and total distances travelled in 10 min were calculated. Data, expressed as percentage of control, were the mean ± SEM of 12 fish larvae per group from 3-time independent experiments. ^##<i>p</i><0.01 <i>versus</i> control group; **<i>p</i><0.01 <i>versus</i> MPTP group (Turkey’s test).</p

SU5416 prevents MPP⁺-induced apoptosis in a concentration-dependent manner.

<p>(A) SU5416, but not VRI, prevented MPP⁺-induced cell death in a concentration-dependent manner. CGNs were treated with SU5416, VRI, EPTU, 7-nitroindazole (7-NI), 1400 W or DMSO (vehicle control) at the indicated concentrations for 2 hours and then exposed to 35 µM MPP⁺. Cell viability was measured by MTT assay at 24 hours after MPP⁺ challenge. (B) SU5416 blocked neuronal loss induced by MPP⁺. CGNs were pre-incubated with or without 20 µM SU5416 and exposed to 35 µM MPP⁺2 hours later. At 24 hour after MPP⁺ challenge, CGNs were assayed with FDA/PI double staining. (C) SU5416 reversed the morphological alteration induced by MPP⁺. CGNs were pre-incubated with or without 20 µM SU5416 and exposed to 35 µM MPP⁺2 hours later. At 24 hour after MPP⁺ challenge, CGNs were assayed with nNOS and Hoechst double staining. (D) The number of apoptotic nuclei with condensed chromatin was counted from representative Hoechst staining photomicrographs and represented as a percentage of the total number of nuclei counted. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *<i>p</i><0.05 and **<i>p</i><0.01 <i>versus</i> MPP⁺ group in (A) or <i>versus</i> control in (D); ^##<i>p</i><0.01 <i>versus</i> MPP⁺ group in (D) (Turkey’s test).</p

Anti-angiogenic effects of SU5416 and VRI in zebrafish.

<p>One dpf Tg<i>(fli-1:EGFP)</i> transgenic zebrafish embryos were treated with SU5416, VRI or DMSO (vehicle control) at the indicated concentrations for 2 days. After treatment, intersegmental-vessel formations were observed under fluorescence microscopy. Deficit of blood vessels was indicated by yellow asterisks.</p

SU5416 reduces the expression of nNOS protein elevated by MPP+ in CGNs.

<p>(A) CGNs were pre-treated with 20 µM SU5416 or DMSO (vehicle control) for 2 hours, and then treated with 35 µM MPP⁺ for various durations as indicated. The total proteins were extracted for Western blot analysis with specific iNOS, nNOS and β-actin antibodies. (B) Statistical analysis of nNOS expression in each treatment group. Data are expressed as the ratio to OD values of the corresponding controls. Data, expressed as percentage of control, were the mean ± SEM of five separate experiments; *<i>p</i><0.05 <i>versus</i> MPP⁺ group at the same time (Turkey’s test).</p

SU5416 increases the number of dopaminergic neurons in MPTP-treated zebrafish larval.

<p>One dpf zebrafish embryos were co-incubated with 200 µM MPTP and 1 µM SU5416 or 0.3% DMSO (vehicle control) for 2 days. After treatment, zebrafish were collected to perform paraffin-embedding, sectioning and immunostaining. (A) Representative picture of immunostaining of zebrafish section. (B) Statistical analysis of the number of TH-positive neurons in each treatment group (n = 12 fish/group). *<i>p</i><0.05 <i>versus</i> MPTP group (Turkey’s test).</p

SU5416 reverses the elevated intracellular NO induced by MPP+ in CGNs.

<p>CGNs were pre-incubated with EPTU, 7-NI or SU5416 at the indicated concentrations for 2 hours, and exposed to 35 µM MPP⁺. Intracellular NO level was measured using DAF-FM diacetate as a probe at 8 hour after MPP⁺ challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **<i>p</i><0.01 <i>versus</i> MPP⁺ group (ANOVA and Dunnett’s test).</p

nNOS depletion abolishes the neuroprotective effects of SU5416 against MPP+-induced neuronal death in PC12 cells.

<p>(A) PC12 cells were transfected with pG418-GFP plasmid (vector), pG418-GFP plasmid encoding nNOS ShRNA (ShnNOS) and pG418-GFP plasmid encoding negative control ShRNA (ShNC). The levels of nNOS and β-actin in the cell lysates were analyzed by Western blotting assay by using specific antibodies. (B, C) nNOS depletion abolished the neuroprotective effects of SU5416 against MPP⁺-induced neuronal death in PC12 cells. PC12 cells transfected with vector, ShnNOS, or ShNC were treated with 20 µM SU5416 for 2 hours and then exposed to 1 mM MPP⁺. Cell viability (B) and cytotoxicity (C) were measured at 24 hours after MPP⁺ challenge by MTT and LDH assays, respectively. Data were the mean ± SEM of three separate experiments; **<i>p</i><0.01 <i>versus</i> control; ^##<i>p</i><0.01 <i>versus</i> MPP⁺ group; ^&<i>p</i><0.05 and ^&&<i>p</i><0.01 <i>versus</i> MPP⁺ vector group (Turkey’s test).</p

SU5416 protects against MPTP-induced TH+ region area decrease in zebrafish.

<p>One dpf zebrafish embryos were co-incubated with 200 µM MPTP and SU5416, VRI or 0.3% DMSO (vehicle control) at the indicated concentrations for 2 days. After treatment, zebrafish were collected to perform whole-mount immunohistochemistry. (A) Representative pictures of whole-mount immunostaining of zebrafish brain from different treatment groups. (B) Magnification of diencephalic area of zebrafish larval (indicated by red bracket in Fig. 2A). (C) Statistical analysis of TH⁺ region area in each treatment group (20 fish embryos per group). Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ^##<i>p</i><0.01 <i>versus</i> control; *<i>p</i><0.05 and **<i>p</i><0.01 versus MPTP group (Turkey’s test).</p