Journal: Science translational medicine

Article Title: Ablation of the stress protease OMA1 protects against heart failure in mice.

doi: 10.1126/scitranslmed.aan4935

Figure Lengend Snippet: Fig. 4. Analysis of ISO response in vitro in neonatal cardiomyocytes. (A) Mitochondrial respirometry analysis of neonatal cardiomyocytes upon different treatments measured by Seahorse and maximum respiration rate (MRR) quantification. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FBS, fetal bovine serum; NAC, N-acetyl-l- cysteine; OCR, oxygen consumption rate. (B) 2′,7′-Dichlorofluorescein diacetate (H2-DCFDA)–mediated ROS quanti- fication in neonatal cardiomyocytes upon different treatments. (C) Fura-2-acetoxymethyl ester (Fura-2 AM)–mediated intramitochondrial calcium quantification in neonatal cardiomyocytes upon different treatments. (D) Tetramethyl- rhodamine methyl ester perchlorate (TMRM)–mediated mitochondrial membrane potential determination in neonatal cardiomyocytes upon different treatments. (E) Analysis of OPA1 processing by Western blot. (F) Analysis of MCU ex- pression by Western blot of mitochondrial lysate. SDHA and core2 are used as loading controls. (G) Kurtosis analysis as indicator of calcium distribution in the cardiomyocytes at the indicated treatments. Basal and final treatments corre- spond to time 0 and time 9 min, respectively. (H) Time-lapse images (40×) at the indicated time showing calcium (Fura-2 AM) and mitochondrial (MitoTracker Deep Red) distribution under different treatments. Scale bars, 20 M. Data in upper and middle panels of (A) are given as means ± SEM and in the rest of graphics as scatter dot plots, and lines are means ± SD. Differences assessed by two-way ANOVA and Sidak’s multiple comparison test (A) or one-way ANOVA and Tukey’s multiple comparison test (B to F). N as described in table S1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used: SDHA (Novex); ATP5B, OPA1, glyceraldehyde-3-phosphate dehydrogenase, core1, core2, and MnSOD (Abcam); MCU and M1CU1 (Sigma-Aldrich); and Tom20 (Santa Cruz Biotechnology).

Techniques: In Vitro, Membrane, Western Blot, Comparison

Journal: Science translational medicine

Article Title: Ablation of the stress protease OMA1 protects against heart failure in mice.

doi: 10.1126/scitranslmed.aan4935

Figure Lengend Snippet: Fig. 7. Heart-specific OMA1 down-regulation protects from hypertrophy induced by TAC. OPA1 processing analysis by Western blot (A) and quantification (B) in isolated mitochondria from heart and liver of mice injected with sh scrambled (shScr) or shOMA1 viral particles. Mitochondria were incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone for the indicated time points. MW, molecular weight; nt, not treated. (C) Des Ao Vel, heart weight/body weight (HW/BW), % EF, and rate of ATP synthesis in WT inbred (C57BL/6JOlaHsd) and outbred (CD1) mice injected with sh shScr or shOMA1 virus and then subjected to TAC. Analyses were performed 3 weeks after TAC surgery. (D) OPA1 processing analysis by Western blot (left) and quantification OPA1 (right) in mitochondrial heart fractions form WT in- bred (C57BL/6JOlaHsd) and outbred (CD1) mice injected with sh scr or sh OMA1 for the indicated conditions. Data are given as scatter dot plots, and lines are means ± SD. Differences assessed by one-way ANOVA and Tukey’s multiple comparison test. N as described in table S1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used: SDHA (Novex); ATP5B, OPA1, glyceraldehyde-3-phosphate dehydrogenase, core1, core2, and MnSOD (Abcam); MCU and M1CU1 (Sigma-Aldrich); and Tom20 (Santa Cruz Biotechnology).

Techniques: Western Blot, Isolation, Injection, Incubation, Molecular Weight, Virus, Comparison

Journal: Resuscitation plus

Article Title: Elevated serum neurologic biomarker profiles after cardiac arrest in a porcine model.

doi: 10.1016/j.resplu.2024.100726

Figure Lengend Snippet: Fig. 4 – Mitochondrial dynamics protein expression and oxidative injury. There is no significant difference in cerebral cortical mitochondrial dynamics via measures of fission (Drp1, Fis1) or fusion (Opa1, Mfn2) relative to GAPDH, or oxidative injury (protein carbonyl, 4-HNE). Western blot protein quantification normalized to GAPDH and presented as relative units.

Article Snippet: Membranes were incubated with antibodies against Opa1 (1:1000, NB110-55290, Novus Biologicals, Littleton CO, USA), Mfn2 (1:2000, ab124773, Abcam, Cambridge, MA, USA), Drp1 (1:1000, 8570S, Cell Signaling Technology, Boston, MA), Fis1 (1:2000, 109561AP, ProteinTech, Rosemont, IL, USA), and GAPDH (1:5000, NB300-327, Novus Biologicals, Centennial, CO, USA) at room temperature for 4 h using iBind technology (ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A Interrogation of ChIP-seq data indicated binding of TAp73α, TAp73β and p53 to the putative OPA1 promoter region. Sequencing read files were obtained from the GEO data set GSE15780, and tracks shown are for the indicated transcription factors at selected genes. B , C Targeted ChIP of TAp73 bound chromatin. RT-qPCR primers were designed in the promoter region of the OPA1 gene (OPA1 ‘A’ and OPA1 ‘B’). Red squares indicate regions enriched for the Trp73 motif (p < 0.001). qPCR was performed to quantify the fold enrichment of the OPA1 promoter region in an IP sample relative to IgG control. Enrichment of MDM2 and SAT2 promoter regions were assayed as positive and negative controls, respectively. qPCR was carried out on 3 independent ChIP experiments and data shown as individual data points ± SD (n = 3). D Representative western blot of mitochondrial fusion proteins in TAp73 KO and WT control. Cells were transfected with either EV or TAp73α expression construct for 24 h. E Densitometry analysis of western blot data shown in (D). Band intensity was calculated for total OPA1 (long and short isoforms) and CDKN1A (positive control). Signal intensity was normalised to loading control and reported relative to WT cells transfected with empty vector (n = 3). *P ≤ 0.05, (n.s.) not significant (Student’s t-test, comparison of indicated condition with WT empty vector control). F RT-qPCR was performed against OPA1 , MFN2 and CDKN1A genes and expression values calculated using the ∆∆Ct method, relative to WT empty vector control. Data shown as mean ± SD (n = 3). *P ≤ 0.05, (n.s) not significant (Student’s t-test, comparison of indicated condition with WT empty vector control).

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Quantitative RT-PCR, Control, Western Blot, Transfection, Expressing, Construct, Positive Control, Plasmid Preparation, Comparison

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: 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.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Control, Western Blot, Construct, Transmission Assay, Injection

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A Immunofluorescence staining performed against the cilia marker Ac-α-tubulin (green) with DAPI nuclear stain in tracheal cross-sections from WT and Trp73 −/− mice (blue). Scale bar = 100 μm. B RT-qPCR for OPA1 mRNA expression in dissociated tracheal epithelial cells. Mouse trachea (n = 3 of each WT and Trp73 −/− ) were pooled together to obtain a sufficient number of cells. C Multiplexed IHC on mouse trachea against Ac-a-tubulin (yellow), OPA1 (red) and DAPI (blue). Cyan arrows indicate MCCs with low OPA1 expression. Scale bar = 30 μm. D Quantification of OPA1 expression in ciliated and non-ciliated cell populations from WT and Trp73 −/− mice. E Images of individual sections of MCCs from WT and Trp73 −/− ciliated epithelium obtained by SBF-SEM. Scale bar = 200 nm. F Mitochondrial length measurements were obtained from (E), including ciliated and non-ciliated cell populations. n ≥ 100 mitochondria from two samples of each genotype. ***P ≤ 0.001, n.s = not significant, in Student’s t-test.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A Trp73 expression data from healthy and COPD individuals from previously described Lung Genomics Research Consortium (LGRC) cohort (GSE47460; Affymetrix array data from n = 157 healthy and n = 220 COPD patients). B Heatmap of Trp73, CDKN1A, OPA1 and FOXJ1 expression in healthy and COPD individuals from the LGRC cohort. The heatmap was generated with the PulmonDB tool using Scipy library in Python, utilising cosine distance and average linkage. Red/blue cells represent positive/negative values. C Row similarity matrix indicating the association between each gene across patient data shown in ( B ). Red shading indicates a positive similarity (measured as 1 - cosine-distance, with similarity values indicated). D , E Expression data showing the correlation between Trp73 and OPA1 ( D ), or Trp73 and CDKN1A ( E ) in COPD patients (GSE47460). Expression values are shown as Log2 fold change for the indicated genes relative to healthy control. The strength of the correlation was calculated using Pearson’s coefficient (r). F , G Analysis of scRNA-seq data from Control and COPD patients. Normalised gene counts were extracted for OPA1 in ciliated cell populations and displayed as mean expression per patient. Control n = 7, COPD n = 12, *P ≤ 0.05 in Student’s t-test. H Correlation plot of mean OPA1 and TP73 expression values per COPD patient. The indicated r value was calculated using Pearson’s coefficient.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: Expressing, Generated, Control

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: TAp73 expression is required for mitochondrial homeostasis in vitro and in the ciliated epithelium in vivo (green nuclei) (left). Conversely, TAp73 ablation leads to decreased OPA1 expression, mitochondrial fission, and impaired mitochondrial function (right). The Trp73 −/− tracheal epithelium maintains a limited expression of FOXJ1 positive cells (purple), indicating additional mechanisms, such as the observed mitochondrial dysfunction drives MCC loss and COPD pathogenesis.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: Expressing, In Vitro, In Vivo

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A list of antibodies used in Western Blotting experiments.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA-TAp73 cloned into the pcDNA3 backbone.

Techniques: Western Blot

Journal: Molecular cell

Article Title: MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

doi: 10.1016/j.molcel.2019.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-Opa1 , Novus Biologicals , NBP1-71656.

Techniques: Recombinant, Protease Inhibitor, FLAG-tag, ATP Bioluminescent Assay, Activity Assay, Western Blot, Autoradiography, Negative Control, Software