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. 2020 Aug 14;11(8):630. doi: 10.1038/s41419-020-02866-3

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© The Author(s) 2020

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. 3 — a Western blotting analysis showed that silibinin induced BNIP3 upregulation and promoted BNIP3 accumulation on mitochondria in a time-dependent manner. b Representative images acquired by confocal microscopy combined with immunocytochemical staining showed that the upregulated BNIP3 induced by silibinin were colocalized with mitochondria. c Silibinin-induced BNIP3 upregulation and its accumulation on mitochondria were both inhibited in the cells transfected with BNIP3 SiRNA. d LDH release assay showed that silibinin-induced glioma cell death was prevented when BNIP3 was knocked down with SiRNA. e Statistical analysis of the intensity of the red fluorescence exhibited by the cells incubated with Mitosox red proved that knockdown of BNIP3 with SiRNA prevented silibinin-induced accumulation of mitochondrial superoxide. f Flow cytometry analysis combined with JC-1 staining showed that knockdown of BNIP3 alleviated silibinin-induced mitochondria depolarization. g Western blotting analysis revealed that silibinin-induced AIF translocation from mitochondria to nuclei was suppressed when BNIP3 was knocked down with SiRNA. h Western blotting proved that silibinin-induced BNIP3 upregulation and accumulation on mitochondria were both inhibited in the presence of 3MA or bafilomycin A1. *p < 0.01 versus control group. The values are expressed as mean ± SEM (n = 5 per group).