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. 2021 Sep 6;11:17705. doi: 10.1038/s41598-021-97162-1

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Modulation of Mortalin levels in primary cultured neurons. (A) Mortalin is down-regulated in neurons upon treatment with rotenone. Rat embryonic primary cortical neurons (DIV 12) were treated or not with 10 nM rotenone (+ rotenone) for 4 h and protein extracts were prepared from mitochondria-enriched fractions. Mortalin amounts were assessed by Western blot, using the mitochondrial chaperone Hsp60 or ß-Actin as loading controls. The graph represents the normalized ratio relative to untreated neurons and is expressed as the mean ± SD of 4 independent experiments. (B) Mortalin loss upon rotenone treatment is due to proteasomal degradation. Neurons (DIV 12) were treated or not with 10 nM rotenone for 4 or 24 h with or without MG132. Neuronal extracts were then analyzed by Western blotting for Mortalin protein quantification. ß-Actin was used as a loading control. The graph represents means ± SD of Mortalin amounts in treated neurons, normalized on control, untreated ones (100%), in 3 independent experiments. (C) Mortalin RNA levels are not affected by rotenone treatment. RNA was extracted from neurons treated or not with 10 nM rotenone for 4 h. qRT-PCR experiments were performed to determine mRNA levels for Mortalin and Rhot2 (Miro2 gene), as well as for Mdh1, a housekeeping gene. The ratios of Mortalin and Rhot2 mRNA levels (normalized on Mdh1) in rotenone treated vs. untreated neurons were calculated and data are presented as means ± SD of 4 independent experiments. (D) Modulation of Mortalin expression by transduction with lentiviral vectors (LV). Overexpression was achieved with a LV bearing an expression cassette for the rat Mortalin gene. Mortalin down-regulation was driven by cell transduction with lentiviral vectors (numbered sh A to D) expressing a set of specific 29-mer shRNA directed against Mortalin (shMortalin), or a control scramble sequence (shScr, Origene TR704140). Different multiplicities of transduction (1, 2, 4) were used to test the percentage of over- or under-expression. WB are presented as cropped parts of full blots displayed as supplemental information. DIV: days in vitro. *: p < 0.05, **p < 0.01, by Mann–Whitney non parametric t-test.