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  Indian J Med Microbiol
 

Figure 2: An autophagy agonist diminishes the neuroprotective effects by reversing autophagy inhibition by SKP-SC-CM. 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) Cell viability was measured by CCK-8 assay, which is presented as the percentage of control cells. (B) Quantitative result of EdU staining. (C) Quantitative analysis of TUNEL-positive cells. (D) Cell proliferation was determined by EdU staining (red). (E) TUNEL staining images. Green: TUNEL-positive cells; blue: Hoechst 33342. SKP-SC-CM inhibited 6-OHDA-induced cell apoptosis, but RAPA increased apoptosis. Scale bars in D and E: 100 μm. (F–H) Western blot analysis of α-syn and TH expression. Relative protein expression was fold to control group. Data are expressed as the mean ± SEM. 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). 3-MA: 3-Methyladenine; 6-OHDA: 6-hydroxydopamine; Con-CM: control conditioned medium; RAPA: rapamycin (autophagy activator); SKP-SC-CM: skin-derived precursor-Schwann cells conditioned medium; TH: tyrosine hydroxylase; α-syn: α-synuclein.

<b>Figure 2: An autophagy agonist diminishes the neuroprotective effects by reversing autophagy inhibition by SKP-SC-CM.</b>
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) Cell viability was measured by CCK-8 assay, which is presented as the percentage of control cells. (B) Quantitative result of EdU staining. (C) Quantitative analysis of TUNEL-positive cells. (D) Cell proliferation was determined by EdU staining (red). (E) TUNEL staining images. Green: TUNEL-positive cells; blue: Hoechst 33342. SKP-SC-CM inhibited 6-OHDA-induced cell apoptosis, but RAPA increased apoptosis. Scale bars in D and E: 100 μm. (F–H) Western blot analysis of α-syn and TH expression. Relative protein expression was fold to control group. Data are expressed as the mean ± SEM. The experiments were repeated five times. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (one-way analysis of variance followed by Tukey’s <i>post hoc</i> test). 3-MA: 3-Methyladenine; 6-OHDA: 6-hydroxydopamine; Con-CM: control conditioned medium; RAPA: rapamycin (autophagy activator); SKP-SC-CM: skin-derived precursor-Schwann cells conditioned medium; TH: tyrosine hydroxylase; α-syn: α-synuclein.