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. 2018 Dec 11;9(12):1189. doi: 10.1038/s41419-018-1229-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — MN9D cells were treated for 12 h with or without 100 μM 6-OHDA or 20 mM 3-MA alone or in combination. a Cells were analyzed by phase-contrast microscopy. Shrunken, phase-bright cells are indicated by white arrows. Scale bar represents 100 μm. b MTT reduction assays were performed to assess cell viability expressed as percentage over the untreated control cells (100%; 60.4 ± 1.3% for 6-OHDA-treated vs. 85.8 ± 0.8% for 6-OHDA plus 3-MA-treated cells). Bars represent the mean ± standard deviation of three independent experiments. ***P < 0.001; n.s. not significant. c Cell lysates were subjected to immunoblotting using anti-LC3, anti-p62 and anti-c-cas-3 antibodies. d−f After normalization against GAPDH, densitometric values of LC3-II, p62, and c-cas-3 signals were expressed as a fold change relative to untreated control value. Bars represent the mean ± standard deviation of three independent experiments (LC3-II, 9.6 ± 0.5 for 6-OHDA-treated vs. 1.9 ± 0.7 for 3-MA-treated vs. 2.0 ± 0.7 for 6-OHDA plus 3-MA-treated cells; p62, 0.3 ± 0.01 for 6-OHDA-treated vs. 2.0 ± 0.1 for 3-MA-treated vs. 0.9 ± 0.1 for 6-OHDA plus 3-MA-treated cells; c-cas-3, 24.6 ± 2.5 for 6-OHDA-treated vs. 0.9 ± 0.04 for 3-MA-treated vs. 1.1 ± 0.2 for 6-OHDA plus 3-MA-treated cells). *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant. g, i Confocal images of cytochrome c (cyto c, green), TOM20 (red) and TUNEL staining (green) in MN9D cells treated with 100 μM 6-OHDA alone or in combination with 20 mM 3-MA were shown. Nuclei were counterstained with Hoechst 33258 (blue). Merged images are shown in the right panel. White arrows indicate condensed or fragmented nuclei. Scale bar represents 20 μm. h The percentage of colocalization between cyto c and TOM20 over total TOM20 was expressed over the untreated control cells (100%). Confocal images of at least 30 randomly selected cells from each of the three independent experiments we used for quantitation. Bars represent the mean ± standard deviation of three independent experiments (25.4 ± 1.1% for 6-OHDA-treated vs. 74.1 ± 0.9% for 6-OHDA plus 3-MA-treated group). **P < 0.01; ***P < 0.001. j The percentage of TUNEL-positive nuclei was assessed from at least 100 randomly selected cells from each of the three independent experiments. Data were shown as the mean ± standard deviation of three independent experiments (0.4 ± 0.4% for untreated vs. 44.0 ± 1.7% for 6-OHDA-treated vs. 0.3 ± 0.2% for 3-MA-treated vs. 12.0 ± 0.6% for 6-OHDA plus 3-MA-treated cells). **P < 0.01; ***P < 0.001; n.s. not significant