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TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis.

In Cell Death Dis on 8 November 2024 by Buckley, N., Craxton, A., et al.

Fig.2.B

TAp73 KO cells display fragmented mitochondria and impaired ETC function.A WT or TAp73 KO H1299 cells were transfected with the indicated plasmids for 24 h and IF carried out against ATP5B (green) with DAPI nuclear counterstain (blue). Cells transfected with HA-TAp73α expression plasmid were stai... more

TAp73 KO cells display fragmented mitochondria and impaired ETC function.A WT or TAp73 KO H1299 cells were transfected with the indicated plasmids for 24 h and IF carried out against ATP5B (green) with DAPI nuclear counterstain (blue). Cells transfected with HA-TAp73α expression plasmid were stained for HA as a transfection control (red). Lower magnification images, scale bar = 10 μm; Callout images, scale bar = 4 μm. B Representative western blot of OPA1 expression following transfection of WT and TAp73 KO cells with pCMW-OPA1 construct. C Quantification of mitochondrial morphology from (A) using Zeiss Intellesis module, trained to segment individual mitochondria. Statistical significance for each condition was calculated using Student’s t-test, comparing with WT EV control (column 1); *p < 0.05, (ns) not significant (n = 3). D Transmission electron micrographs of mitochondrial morphology from WT and TAp73 KO cells. Scale bar = 100 nm. E Mitochondrial length measurements obtained from (D). ****p < 0.0001 in Student’s t-test. A minimum of 100 mitochondria were measured from n = 3 independent biological replicates. F, G Mitochondrial stress test performed on Seahorse XFe96 analyser. Canonical mitochondrial inhibitors injected sequentially as labelled (Oligomycin = 2 μM, FCCP = 500 nM, Antimycin A/ Rotenone = 2 μM). The indicated mitochondrial stress test parameters were calculated from OCR data. Data were corrected for non-mitochondrial OCR, normalised to cell number, and are shown as mean ± SD (n = 3). *P ≤ 0.05 and **P ≤ 0.01 in Student’s t-test relative to WT control. H Western blot of the indicated ETC subunits in wild-type and TAp73 KO cells, obtained using OXPHOS antibody cocktail. I qPCR against mt-CO2, expressed relative to expression of nuclear encoded β2-microglobulin. Relative expression was calculated using the ΔΔCt method and expressed as a percentage of wild-type control (n = 2 biological replicates, each tested in triplicate). n.s not significant in Student’s t-test. less

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Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status.

In Sci Rep on 31 May 2024 by Marques, P., Burillo, J., et al.

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Resveratrol induces both autophagic and mitophagic fluxes in MIN6 Scr pancreatic beta cells. (A) Representative western blots of different mitophagy markers in MIN6 Scr and MIN6 Tsc2 shRNA pancreatic beta cells treated or not with resveratrol at 50 µM using a time-course analysis. (B) The graphs ... more

Resveratrol induces both autophagic and mitophagic fluxes in MIN6 Scr pancreatic beta cells. (A) Representative western blots of different mitophagy markers in MIN6 Scr and MIN6 Tsc2 shRNA pancreatic beta cells treated or not with resveratrol at 50 µM using a time-course analysis. (B) The graphs represent the densitometric study showing the mean and SD; n = 4 independent experiments. One-way ANOVA was performed with Tukey's multiple comparisons test as post hoc. *p ≤ 0.05, **p ≤ 0.01 MIN6 Tsc2 Scr vs MIN6 Tsc2 shRNA; #p ≤ 0.05 MIN6 treated with resveratrol 50 µM vs control. (C) Transmission electronic microscopy (TEM) images obtained from MIN6 Tsc2 Scr and Tsc2 shRNA treated or not with resveratrol 50 µM. The bar represents 0.2 µm. *p ≤ 0.05 and **p ≤ 0.01 comparing control versus resveratrol in the MIN6 Scr cells. (D) Representative western blots for the study of autophagic and mitophagic fluxes in MIN6 Scr and Tsc2 shRNA pancreatic beta cells treated with either nicotinamide (5 mM) or resveratrol (50 µM) in the presence or in the absence of a pre-treatment with chloroquine (CQ) for 24 h. The graphs correspond to the densitometric analysis of all the blots showing the mean ± SD, n = 3. One-way ANOVA was performed with Tukey's multiple comparisons test as post hoc. **p ≤ 0.01, ***p ≤ 0.001 MIN6 Tsc2 Scr vs MIN6 Tsc2 shRNA. less

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Mitochondrial Homeostasis Regulating Mitochondrial Number and Morphology Is a Distinguishing Feature of Skeletal Muscle Fiber Types in Marine Teleosts.

In Int J Mol Sci on 26 January 2024 by Li, B., Wang, H., et al.

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Slow-twitch muscles have higher mitochondrial fusion and fission rates compared to fast-twitch muscles, n = 3. (A) Expression of genes associated with mitochondrial fusion, including mfn2 and opa1, between two types of muscle. (B,C) Protein expression of MFN2 and OPA1 were evaluated through Weste... more

Slow-twitch muscles have higher mitochondrial fusion and fission rates compared to fast-twitch muscles, n = 3. (A) Expression of genes associated with mitochondrial fusion, including mfn2 and opa1, between two types of muscle. (B,C) Protein expression of MFN2 and OPA1 were evaluated through Western blotting and normalized to GAPDH protein. The mean plus standard deviation of 3 biological replicates is shown. (D,E) Representative TEM images and quantification of mitochondrial area of two muscle types. (F) Expression of genes associated with mitochondrial fission, including Drp1, Dnm2, and Mff, in two types of muscle. less

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Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Clonogenic Cell Survival in Human Microvascular Endothelial Cells.

In Cells on 23 December 2023 by Wang, L., Rivas, R., et al.

Fig.7.A

The impact of doses on mitochondrial fusion and fission machinery in irradiated HMEC-1. The examination was conducted on day 4 post-IR. (A,C) 1 Gy and 0.5 Gy upregulated Mfn1 expression, as determined by Western blot analysis. However, no differences in Mfn2 expression were observed across all do... more

The impact of doses on mitochondrial fusion and fission machinery in irradiated HMEC-1. The examination was conducted on day 4 post-IR. (A,C) 1 Gy and 0.5 Gy upregulated Mfn1 expression, as determined by Western blot analysis. However, no differences in Mfn2 expression were observed across all doses tested, as shown in panels (A,D). (A,E) High doses decreased Opa1 expression. (B,F) There were no differences in Drp1 expression observed across all doses tested. There were three samples per radiation dose, and the statistical analysis conducted using one-way ANOVA and Dunnett’s multiple comparisons test revealed significant differences between irradiated cells and control cells, denoted by * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. The mitochondrial loading control in Western blot analyses was the voltage-dependent anion channel (VDAC), whereas the cytosol loading control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The abbreviations used in the figures are as follows: Mfn1 represents mitofusin 1; Mfn2 represents mitofusin 2; Opa1 represents optic atrophy 1; and Drp1 represents dynamin-related protein 1. Data are expressed as mean + SEM. less

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Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson's disease mouse model.

In Nat Commun on 13 November 2023 by Fang, T. Z., Sun, Y., et al.

Fig.1.C

Generation and characterization of Usp30 KO mice.a Schematic of gene targeting to generate the Usp30 KO mice. b Bar graph shows the levels of Usp30 gene expression in different tissues in Usp30 WT and KO mice (n = 5 for brain, n = 4 for testis, n = 3 for all other tissues). Error bars represent m... more

Generation and characterization of Usp30 KO mice.a Schematic of gene targeting to generate the Usp30 KO mice. b Bar graph shows the levels of Usp30 gene expression in different tissues in Usp30 WT and KO mice (n = 5 for brain, n = 4 for testis, n = 3 for all other tissues). Error bars represent mean ± s.d. c Representative Western Blot images of OPA1, beta-actin and USP30 in the cortex of Usp30 WT and KO male mice. The experiment was repeated twice independently. d Estimated and observed numbers of WT, Usp30 heterozygous (Het) and Usp30 homozygous knockout (KO) mice in the offspring of heterozygous Usp30 breeders. e Survival curve of WT and Usp30 KO mice. f schematic image showing the working mechanism of mito-QC reporter protein for assessing the mitophagy signal in cells. g Representative fluorescence images show the mito-QC fluorescence signal (mCherry-red, GFP-green), and dopaminergic neurons (TH, blue) in the SNpc of mito-QC and mito-QC/Usp30 KO male mice. Dashed white inlets were enlarged in right panels showing the details of mCherry only puncta (mitophagy puncta) in the DA neurons. Scale bar, 10 µm. h Quantification of mitophagy puncta in individual dopaminergic neurons of the SNpc (n = 13 for USP WT male mice, n = 14 for Usp30 KO male mice, 5–10 neurons per mouse). WT and Usp30 KO in the bar graph represent mito-QC and mito-QC/Usp30 KO, respectively. Significance determined by unpaired, two-tailed Student’s t test. Error bars represent mean ± s.d.; *P < 0.05. Acronyms: WT wild type, GT gene-trap, KO knock out, loxP locus of X-over P1 site, FRT flippase recognition target, SA splice acceptor, pA polyA tail, NEO neomycin resistance cassette, LacZ β-galactosidase. Source data are provided as a Source Data file. less

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