Dopaminergic neuronal loss accompanied by the deprivation of netrin-1. (A) Co-immunofluorescence staining analysis upon DCC-4Fbn or control IgG treatment primary dopaminergic neurons conducted by TH/MAP2, MAP2/NTN1, and MAP2/pS129 (Scale bar, 50 μm). (B) Quantification bar graph of TH, MAP2, NTN1, and pS129 immunoreactive positive dopaminergic neuron cell numbers. Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 4 each group. **P < 0.01. (C) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in primary dopaminergic neurons after control IgG and DCC-4Fbn treatments (Scale bar, 40 μm). (D) DNA fragmentation index (bar graph) expressed as a percentage of TUNEL positive dopamine (DA) neurons out of the total number of MAP2-positive neurons. N = 3 each group. Error bars represent the mean ± SEM. Statistical significance was conducted using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. **P < 0.01. (E) Immunoblotting assay of netrin-1, TH, MAO-B, cleaved caspase-3, α-Syn pS129 and β-actin levels in control IgG (N = 3) vs DCC-4Fbn treated cell lysates (N = 3). Band intensity quantification bar graph (right). (F) Immunoblot of SH-SY5Y cell lysates with treated neurotoxin MPP⁺ in a dose-dependent manner (1-3 mM). Relative band intensity were calculated from the band densitometry software (Image J). Quantification of band intensity (right). N = 3 independent immunoblot assay. Error bars represent the mean ± SEM. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01; N.S., not significant.
