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: 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

Journal: International Journal of Molecular Sciences

Article Title: CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy

doi: 10.3390/ijms26136364

Figure Lengend Snippet: Characterisation of c.2708_2711del HEK293 cell line. ( A ) Sanger sequencing results from the heterozygous and homozygous clones obtained after CRISPR edit, with reference WT sequence above. The bases targeted for deletion are highlighted in purple. Exon 27 is overlined in black. ( B ) Quantification of OPA1 transcript levels in edited cells compared to WT through qPCR. Bars indicate means ± SD. A total of 50 µg/mL of emetine was added to (+) cell samples for 4 h prior to RNA extraction. GAPDH and Actin were used as reference genes. p values were determined through two-way ANOVA. Values between untreated (blue bars) and emetine-treated samples (red bars) showed no significant difference in two-way ANOVA. ( C ) Western blots showing OPA1 protein, both l- and s-forms (upper and lower bands, respectively). ( D ) Quantification of total OPA1 levels (sum of OPA1 l- and s-forms) shows a relative decrease in edited cells. Actin stain used as a loading control. A total of 50 µg/mL of emetine was added to (+) cell samples for 4 h prior to cell lysis. A total of 8 µg of protein was loaded for each sample. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: The membranes were stained with rabbit anti-OPA1 primary antibody (R&D systems, Minneapolis, MN, USA) at a dilution of 1:2000 and HRP-conjugated mouse anti-β-Actin primary antibody (Abcam, Cambridge, UK) at a dilution of 1:25,000.

Techniques: Sequencing, Clone Assay, CRISPR, RNA Extraction, Western Blot, Staining, Control, Lysis

Journal: International Journal of Molecular Sciences

Article Title: CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy

doi: 10.3390/ijms26136364

Figure Lengend Snippet: OPA1 transcript and protein upregulation mediated by dCas9–VPR. ( A ) Diagrammatic representation of the position of gRNAs in relation to OPA1’s transcription start site. ( B ) Relative OPA1 mRNA transcript upregulation measured through qPCR, with ACTIN and GAPDH used as reference genes. ( C ) OPA1 protein expression detected through Western blot and ( D ) quantification of the relative OPA1 protein level using actin immunoreactivity as a reference protein. A total of 8 µg of protein was loaded for each sample. n = 3. p values were determined through one-way ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: The membranes were stained with rabbit anti-OPA1 primary antibody (R&D systems, Minneapolis, MN, USA) at a dilution of 1:2000 and HRP-conjugated mouse anti-β-Actin primary antibody (Abcam, Cambridge, UK) at a dilution of 1:25,000.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy

doi: 10.3390/ijms26136364

Figure Lengend Snippet: Maintenance of OPA1 isoform ratios in CRISPRa-treated cells. ( A ) Diagrammatic representation of OPA1 isoforms with alternatively spliced exons marked in red. Two sets of RT-PCR primers were used to separate isoforms containing and excluding exon 5b, with these primers marked by the arrows. The product size for each primer was marked accordingly. ( B ) RT-PCR isoform products separated on a 2% agarose gel. Black arrows indicate the bands detected ( C , D ) Each band was quantified as a percentage of the total transcripts for each amplicon. n = 3. Only isoform 5 showed any significant differences between the conditions and is marked in blue. p values were determined through two-way ANOVA. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Article Snippet: The membranes were stained with rabbit anti-OPA1 primary antibody (R&D systems, Minneapolis, MN, USA) at a dilution of 1:2000 and HRP-conjugated mouse anti-β-Actin primary antibody (Abcam, Cambridge, UK) at a dilution of 1:25,000.

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification