Fig. 3: ANP induces β-catenin stabilization and nuclear translocation and is neuroprotective in primary cultures of DA neurons from mouse brain.

A Confocal microscopy images showing the intracellular distribution of β-catenin (red hue) in control and ANP treated DA neurons (TH positive; green hue) after 24 h; double immunofluorescent staining β-catenin/TH. Nuclei were visualized by Hoechst counterstaining (blue hue). Merged images with differential interference contrast (DIC), used for visualizing cell morphology, are also shown. Arrows point to β-catenin positive nuclei in TH positive cells. Bars 25 µm. B Quantitative evaluation of the percentage of TH positive cells exhibiting nuclear β-catenin, in untreated and ANP treated cultures. Data were obtained by counting a minimum of 300 cells/samples and results showed are the mean ± SD from three independent experiments (n = 3). Significance vs. untreated control (two-tailed Student’s t test): ^*p < 0.05. C WB and densitometric analysis of total and phosphorylated β-catenin (pβ-catenin^T41/S65), TH, Nurr-1, and DJ-1 levels in total cell lysates from ANP treated cells, compared to the untreated control, after 24 h of culture. In the densitometric analysis, values were normalized to β-actin, or vs. the total levels of β-catenin for the phosphorylated form. Results are the mean ± SD from three independent experiments (n = 3). Significance vs. control (two-tailed Student’s t test): ^*p < 0.05; ^**p < 0.01. D Phase contrast microscopy of primary DA neurons exposed to 25 µM of 6-OHDA for mimicking the neurodegeneration of PD. To assess the neuroprotective ability of NPs against 6-OHDA induced cytotoxicity, cells were pre-treated with 100 nM of the hormone 30 min prior to 6-OHDA addition to the cell culture medium. Bars 100 µm.