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. 2021 Jul 9;12:4220. doi: 10.1038/s41467-021-24469-y

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© The Author(s) 2021

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. 6 — a GFP-tagged Prok2-AAV is injected into mouse brain at 1 week before CCI. Dual-labeled immunofluorescence staining with Prok2-eGFP (green) and NeuN (red) is used to test the efficiency of AAV transfection in neurons. Scale bar is 100 μm (left) and 30 μm (right). b Increased brain tissue Prok2 expression is detected by western blot at 7 days after i.c.v. injection of GFP-tagged Prok2-AAV. GAPDH is used as control. c, d AAV-shFbxo10 carrying luciferase is injected into mouse brain tissue. Cri Maestro In-vivo Imaging Systems is used to screen for successful transfection. Fbxo10 knockdown in brain tissue is confirmed by western blot assays. GAPDH is used as control. e–g 2 days after CCI, protein expression of Gpx4 and Acsl4 proteins is examined by western blot analysis. Fer-1(1 mg/kg per day) is given i.p. once daily for 7 days before CCI and continued until euthanasia. While Acsl4 levels increases. Gpx4 levels do not change after CCI. AAV-Prok2 administration increases Gpx4 but decreases Acsl4 expression, which is blocked by Fbxo10 knockdown after CCI. Fer-1 administration suppresses CCI-induced increases in Acsl4 levels and alleviates AAV-shFbxo10-induced decrease in Gpx4 expression. Data are presented as mean values ± SD (n = 3 mice per group). h and i Representative T2 weighted MR images showing lesion volume in mouse brain after CCI in different experimental groups. While AAV-Prok2 transfection reduces lesion volume, co-transfection of AAV-Prok2 and AAV-shFbxo10 abolishes this effect. Administration of Fer-1 on the other hand decreases lesion volume in CCI mice expressing AAV-Prok2 and AAV-shFbxo10. Data are presented as mean values ± SD (n = 4 mice per group). j Mitochondrial morphology under different conditions is evaluated by electron microscopy. CCI-induced shrunken mitochondria and rupture of OMM, ferroptosis-related morphological changes of mitochondria (red arrow), are prevented by AAV-Prok2. k Immunostaining is used to examine the spatial distribution Gpx4 and Acsl4 expression in pericontusional area and shows similar treatment effect that is seen in western blot analysis observed in panels f, g. l, m Cell death response in the pericontusional area is quantified using TUNEL. Data are presented as mean values ± SD (n = 4 mice per group). For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file.