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Results: 4

1.
Figure 3

Figure 3. Effect of OLE on Aβ oligomer production.. From: Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing A?42 by Reducing Plaque Load and Motor Deficit.

Representative dot blot of (A) total Aβ (WO2) and (C) Aβ oligomers (A11) in CL2006 transgenic worms fed with vehicle or with 50 µM OLE at L2 larval stage. Equal amounts of proteins from worm lysates (5 µg) were spotted in triplicate. Total proteins on the blotted membranes were stained using a 0.1% Red Ponceau solution and were used to normalize the immuno-specific signal for protein loading. The mean volume of the Aβ-reactive and Red Ponceau-dyed spots was determined using the Progenesis SameSpots software (Nonlinear Dynamics, UK). Immunoreactivity of WO2 (B) or A11 (D), from three independent experiments (n = 9), was expressed as the mean volume of the immunoreactive band/volume of Ponceau-dyed proteins±SD.*p<0.05 vs. vehicle (Student’s t-test).

Luisa Diomede, et al. PLoS One. 2013;8(3):e58893.
2.
Figure 4

Figure 4. Effect of OLE on oxidative stress in CL2006 transgenic C. elegans strain.. From: Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing A?42 by Reducing Plaque Load and Motor Deficit.

Egg-synchronized CL2006 transgenic worms were placed at 16 °C on E. coli for 48 h and then fed, at L2 larval stage, with vehicle or with 50 µM OLE for 48 h (100 µl/plate). Tetracycline and NAC were used as positive controls for antioxidant activity. Worms were then collected in 1.6 ml 1% Tween 20 in PBS and the NBT assay was done as described in Materials and Methods. Results are calculated as absorbance units/mg of protein (n = 100 worms/group, three independent experiments) and expressed as % of control, i.e. the percentage of superoxide produced by drug-treated CL2006 worms, considering 100% that produced by vehicle treated ones.**p<0.01 vs. vehicle-treated CL2006 worms, according to One-way ANOVA and Bonferroni’s post test analysis.

Luisa Diomede, et al. PLoS One. 2013;8(3):e58893.
3.
Figure 1

Figure 1. Effect of OLE on Aβ-induced paralysis in CL2006 transgenic C. elegans strain.. From: Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing A?42 by Reducing Plaque Load and Motor Deficit.

(A) Diagram illustrating the paralysis assay showing when the drug was administered and when the paralysis assay was scored. (B–C) Dose-response effect of OLE on the paralysis induced by Aβ expression in CL2006 and CL802 transgenic worms. Egg-synchronized worms were placed at 16 °C on fresh NGM plates seeded with OP50 E. coli and, at L2 (B) or L3 (C) stage, were fed with OLE (12.5–500 µM). The number of paralyzed worms was scored 48 h or 24 h after treatment (at L4 larval stage) for L2- and L3-treated worms, respectively. Data are shown as percentage±SE of paralyzed worms to vehicle treated ones (n = 100 worms/group, 3 independent assays). (D) Percentage of paralyzed worms fed with OLE. CL2006, CL802 and N2 worms, cultured as above, were fed 50 µM OLE at L1 or L2 and 500 µM at L3. Tetracycline at 50 µM was administered at L3 as positive control. The number of paralyzed worms was scored at L4 larval stage. Data are shown as percentage±SD of paralyzed worms to vehicle treated ones (n = 100 worms/group, 3 independent assays). °° p<0.01 vs. CL802, **p< 0.01 vs CL2006 worms fed with vehicle (One-way ANOVA test), and +p<0.01 vs. CL2006 worms fed with 50 µM OLE at L2 (Student’s t test).

Luisa Diomede, et al. PLoS One. 2013;8(3):e58893.
4.
Figure 2

Figure 2. OLE reduces amyloid deposition and extends the life-span of CL2006 transgenic C. elegans strain.. From: Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing A?42 by Reducing Plaque Load and Motor Deficit.

Egg-synchronized CL2006 worms were placed at 16 °C on fresh NGM plates seeded with OP50 E.coli and, at L2 larval stage, were fed with vehicle or with 50 µM OLE for 48 h. (A) At 120 h of age, corresponding to day 1 of adult age, worms were stained with X-34 dye. The staining of amyloid plaques on whole-mount and fixed samples was visualized at short wavelength excitation. Scale bar 20 µm. Arrows in red indicate amyloid deposits. (B) Amyloid deposits in the anterior area of worms fed with vehicle (n = 20) and OLE (n = 20), quantified by counting the number of X-34 positive spots. **p<0.01 vs. CL2006 worms fed with vehicle (Student’s t test). (C–D) Kaplan-Meier survival curves of (C) CL2006 and (D) CL802 worms fed with vehicle or with 50 µM OLE at L2 larval stage. The number of paralyzed worms (considered dead) was scored 48 h after treatment, at L4 larval stage (day 0 in graph) and every consecutive day, until all worms were dead. Survival is expressed as a percentage of the initial population. Plots are representative of three independent experiments (n = 30 worms/group).

Luisa Diomede, et al. PLoS One. 2013;8(3):e58893.

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