Figure 3: RAPA weakens the protective effects of SKP-SC-CM by activating autophagy in 6-OHDA-induced SH-SY5Y cells. SH-SY5Y cells were pretreated with SKP-SC-CM, RAPA or 3-MA for 4 hours and then treated with 6-OHDA (50 μM) for 12 hours. (A–C) Western blot analysis of LC3B and p62 expression. Data are expressed as the mean ± SEM, normalized to the control group. The experiments were repeated five times. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). (D, E) Double immunofluorescence staining of LC3B (green, Alexa Fluor-488) or p62 (green, Alexa Fluor-488) and TH (red, Alexa Fluor-647). (F) Autophagic vesicles or autophagosomes in cells were identified by transmission electron microscopy. White triangle: autophagosome; N: nucleus. Autophagic vesicle magnification is presented in the white square. Scale bars: 50 μm in D, E; 20 μm in F. 3-MA: 3-Methyladenine; 6-OHDA: 6-hydroxydopamine; Con-CM: control conditioned medium; LC3B: microtubule-associated protein 1 light chain 3; p62: sequestosome 1; RAPA: rapamycin (autophagy activator); SKP-SC-CM: skin-derived precursor-Schwann cells conditioned medium.