Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 is upregulated in ischemic heart and dysregulates mitochondrial pathways in cultured cardiomyocytes. A . Graph showing transcript levels of ventricular Letm1 in human patients ( n = 5 and ICM n = 4) ( B ) and mouse model (Sham n = 5, LAD n = 3) of ischemic cardiomyopathy compared to respective non-failing hearts. C . Protein levels of ventricular Letm1 in human patients (NF n = 5, ICM n = 10) and mouse model of ischemic cardiomyopathy (Sham n = 6, LAD n = 5) compared to respective non-failing hearts. Its densitometry is depicted in D and E , respectively. Transcript levels of ventricular Letm1 in human patients (NF n = 4, HCM n = 3) ( F ) and mouse model ( n = 6 ORAB n = 6) ( G ) of cardiac hypertrophy compared to respective non-failing hearts. H . Protein levels of ventricular Letm1 in human patient and mouse model of cardiac hypertrophy compared to respective non-failing hearts(NF n = 5, HCM n = 7, Sham n = 7, ORAB n = 6). Its densitometry is depicted in I and J , respectively. K . Heatmap of differentially expressed genes upon Letm1 expression compared to LacZ control. L . Volcano plot highlights some of the significantly up- and down-regulated genes upon Letm1 expression compared to LacZ control. NF: non-failing; HCM: hypertrophic cardiomyopathy; ICM: Ischemic cardiomyopathy; LAD: left anterior descending artery ligation; ORAB: O-ring aortic bandin; GO: Gene ontology, Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Cell Culture, Expressing, Control, Ligation, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 dysregulates OXPHOS gene expression and protein abundance in cultured cardiomyocytes. A - G . Transcript level detection of genes involved in oxidative phosphorylation complexes upon Letm1 expression. H . Immunoblot of oxidative phosphorylation (OXPHOS) complexes detected in total protein lysates and its desitometric analysis in ( I ). J - M Transcript level alteration of mitochondrial genes in iPS derived cardiomyocytes post elevated letm1 levels as compared to LacZ control. N . Immunoblot of oxidative phosphorylation (OXPHOS) complexes detected in total protein lysates from iPSC-derived cardiomyocytes and its densitometric analysis in ( O ). Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Gene Expression, Quantitative Proteomics, Cell Culture, Phospho-proteomics, Expressing, Western Blot, Derivative Assay, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 overexpression impairs mitochondrial bioenergetics and functional integrity in cardiomyocytes. A . Results from mitochondrial function assessment using seahorse metabolic assay (Mito Stress Test) performed in NRVCMs indicating oxygen consumption rate (OCR) for mitochondrial respiration ( B ) ATP production, ( C ) maximal respiration, ( D ) proton leak, and ( E ) coupling efficiency ( F ). Spare respiratory capacity, G . Oxygen consumption rate in the mito stress test assay. H . Oxidative phosphorylation calculated from the ATP rate assay detecting reduced levels upon Letm1 elevation, I . ECAR upon Letm1 elevation, J . ATP production graph plotted using measurements from ATP rate assay to calculate ATP obtained from mitochondrial vs. glycolysis pathway. K . Graph showing the results from glucose uptake assay for NRVCMs either expressing Letm1 or LacZ control. Immunoblot ( L ) and its densitometry ( M ) depicting Cpt1a levels in NRVCMs expressing Letm1 compared to LacZ control. N . Graph showing transcript levels of Glut1 in NRVCMs expressing Letm1 compared to LacZ control. O . Proton efflux rate from the glycolytic rate assay upon Letm1 overexpression. P . Elevated levels of basal glycolysis upon Letm1 overexpression as compared to control. Q . Increase in compensatory glycolysis upon Letm1 overexpression. R . Substrate utilization assay revealed glucose dependency as the main substrate utilized for ATP generation in Letm1 overexpressing cells. S . Increased % of glucose dependency of Letm1 overexpressing cells as compared to LacZ condition. T . Decreased % of fatty acid utilization of Letm1 overexpressing cells as compared to LacZ condition. ECAR; Extracellular acidification rate, OCR; oxygen consumption rate, PER; proton efflux rate. n = 3 for all experiments. Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Over Expression, Functional Assay, Metabolic Assay, Phospho-proteomics, Expressing, Control, Western Blot, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Elevated levels of Letm1 disrupts electrophysiology and contractility in cardiomyocytes. A Representative graphs of action potential measurements of cardiomyocytes expressing Letm1 as compared to LacZ control. B Graph plotted for action potential duration at 50% ( B ) and 90% ( C ) repolarization. D . L-type Calcium current density of cells with Letm1 vs. LacZ. E . Representative Calcium current measurements of Letm1 vs. LacZ expressing cells. F . Calculated Calcium current density. G . Sustained Potassium current of cells with Letm1 vs. LacZ. H . Representative sustained Potassium current measurements of Letm1 vs. LacZ expressing cells. I . Calculated Potassium current density. APD: action potential duration, n = 3. Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 expression disrupts contractility in cardiomyocytes. A - H . Transcript levels of genes involved in Potassium, sodium and Calcium ion transport in Letm1 expressing or LacZ expressing control NRVCMs. I .- L . Transcript levels of genes involved in Potassium, sodium and Calcium ion transport in Letm1 expressing or LacZ expressing control in NRVCMs under hypoxia condition as compared to normoxia condition. M . Representative images of the peaks obtained from cardiomyocyte contractility assay. N . Cardiomyocyte contractility calculated from the obtained data. O . Relaxation velocity of cardiomyocytes upon Letm1 expression. n = 3. Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 overexpression alters mitochondrial ultrastructure and sarcomeric organization in adult cardiomyocytes. A Representative electron microscopy images of Letm1 overexpressing adult cardiomyocytes as compared to LacZ expressing control. In overviews (10 μm scale), cardiomyocytes with Letm-1 overexpression feature large vacuoles typically at the Z-level of sarcomeres (arrowheads). Images at higher magnification (1 μm scale) demonstrate disturbance of the Membrane-system in Letm1 condition as compared to LacZ control (T, sarcoplasmic T-tubule system), containing multivesicles (MV) and cellular content, including mitochondria (M), reminiscent of autophagosomes. The detailed view (100 nm scale) in the upper panel depicts defective mitochondria inside a vesicle (M*) and in the lower panel healthy mitochondria and multivesicular body (MV) between two regular mitochondria (M). S; sarcomere, M; mitochondria, M*; defective mitochondria, T; T tubules. B Reduced mitochondrial mass was observed in Letm1 overexpressing cells as compared to control cell. C Mitochondrial number was observed to be increased in count upon Letm1 overexpression. D Sarcomeric length was significantly reduced in cells overexpressing Letm1, E , F . Quantitative mitochondrial DNA copy number was determined using realtime PCR of mitochondrial to nuclear genes at DNA level. n = 3. Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Over Expression, Electron Microscopy, Expressing, Control, Membrane, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 overexpression activates autophagy but impairs lysosomal clearance. A . Immunoblots indicating different markers for AMPK-ULK1 signaling pathway and autophagy in Letm1 overexpressing NRVCMs as compared to LacZ control condition along with their respective densitometric analysis in ( B - H ). I . Increased transcript level of Parkin upon Letm1 elevation. J . Representative images of Tandem LC3-GFP-RFP imaging in cells with increased Letm1 in combination with BafilomycinA1 treatment and its respective analysis in K , L . M . Immunoblots indicating p62 and LC3 levels upon Letm1 overexpression as compared to LacZ control in presence and absence of Bafilomycin A1 treatment and its respective densitometric analysis in N , O respectively. Baf; BafilomycinA1. n = 3. Statistical significance is calculated by two-tailed Students’ t-test or 2-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Over Expression, Western Blot, Control, Imaging, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Elevated levels of Letm1 drives mitochondrial dysfunction and cardiomyocyte stress-mediated apoptosis in cultured cardiomyocytes

doi: 10.1186/s12964-025-02378-7

Figure Lengend Snippet: Letm1 overexpression impairs cardiomyocyte survival, and stress response via mitochondrial dysfunction and increased apoptosis. A . Representative immunofluorescence images of cardiomyocytes stained with α-actinin and DAPI (scalebar 50 μm) for cell surface area measurement and it analysis is shown in ( B ). Transcript levels of Nppa ( C ) and Nppb ( D ), in cardiomyocytes with either Letm1 or LacZ overexpression. E . Representative immunofluorescence images of cardiomyocytes stained with TMRE (Scalebar 50 μm) for mitochondrial membrane potential measurements depicted in ( F ). G . Cardiomyocyte cell viability detected by MTT assay. H . Immunoblots detecting Caspase 3 and its cleaved fragment as well as Caspase 7 and its cleaved fragment. Its densitometry measurements are depicted in I , respectively. n = 3 for every experiment. Statistical significance is calculated by two-tailed Students’ t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant

Article Snippet: Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, including: Letm1 (1:1000, Santa Cruz Biotechnology SC271235), OXPHOS antibody cocktail rodent (1:5000, Abcam 110413), Caspase 3 (1:1000, Cell Signaling Technology, 9662), Caspase 7 (1:1000, Cell Signaling Technology 9492), α-actinin (1:2000, Sigma-Aldrich, A7732), Cpt1a (1:1000, Proteintech, 66039-1-Ig), ULK1 (1:1000, CST, 8054), pULK1 (1:1000, CST, 5869), AMPK (1:1000, CST, 2534), DRP1 (1:1000, CST, 5391), pDRP1 ser 616 (1:1000, 4494), LAMP2 (1:1000, GTX635790), BNIP3L (1:1000, GTX64447), p62 (1:1000, CST, 5114), LC3 (1:1000, CST, 2775).

Techniques: Over Expression, Immunofluorescence, Staining, Membrane, MTT Assay, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: A549 cells were infected with GFP (Ad-GFP), LETM1 (Ad-LETM1), or without adenovirus (Control) for 48 h. (A) Mitochondria proteins were analyzed by immunoblotting with the indicated antibodies [mitochondrial-encoding respiration chain proteins: NADH: ubiquinone oxidoreductase 6 (ND6), a complex I subunit; succinate dehydrogenase complex subunit A (SDHA), a complex II subunit; and cytochrome c , complex III subunit; COXIV, a complex IV subunit and another mitochondrial protein: apoptosis-inducing factor (AIF), heat shock protein 60 (HSP60), and voltage-dependent anion channel (VDAC)]. (B) The bands-of-interest were further analyzed by densitometry. (C) Mitochondrial ATP was measured by luciferase assay. (D) Expression level of phospho-AMPK at Thr172 and AMPK proteins. (E) The bands-of-interest were further analyzed by densitometry.(F) A549 cells were transfected with siRNA LETM1 in time-dependent manner and then Western blot analysis of phospho-AMPK at Thr172. (G) Cells were fixed and immunostained with anti-phospho-AMPK (Thr172) antibody and then visualized on a confocal microscope. Each bars represent mean±SE (n = 3), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Infection, Control, Western Blot, Luciferase, Expressing, Transfection, Microscopy

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: The 9 weeks K -ras LA1 mice were exposed to aerosols containing adenovirus-LETM1 twice a week for total 4 weeks. Lung tissue homogenates were subjected to western blot analysis. (A) Expression level of LETM1, phospho-AMPK at Thr172 and AMPK proteins. (B) The bands-of-interest were further analyzed by densitometry. (C) Expression level of Akt, phospho-Akt at Ser473, phospho-Akt at Thr308, mTOR and phospho-mTOR at Ser2448 proteins. (D) The bands-of-interest were further analyzed by densitometry. Each bar represents mean±SE (n = 8), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values. (E) The effect of only siRNA AMPK or with infected adenovirus-LETM1 and protein expression level of AMPK and phospho-Akt at Thr 308.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Western Blot, Expressing, Control, Infection

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: A549 cells was infected with GFP (Ad-GFP), LETM1 (Ad-LETM1), or without adenovirus (Control) for the indicated times. (A) Cell cycle distribution was determined by FACS. (B) Percentage of G1/G0 phase cell population. (C) Expression of LETM1 Protein for different times. (D) Expression of p21, cyclinE, cyclinD1, CDK4, p27, phospho-p53 at Ser15 and p53 proteins. (E)The bands-of-interest were further analyzed by densitometry. Each bars represent mean±SE (n = 3), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Infection, Control, Expressing

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: The 9 weeks K -ras LA1 mice were exposed to aerosols containing adenovirus-LETM1 twice a week for total 4 weeks. Lung tissue homogenates were subjected to western blot analysis. (A) Expression of p21, cyclinE, cyclinD1, CDK4, p27, phospho-p53 at Ser15 and p53 proteins. (B) The bands-of-interest were further analyzed by densitometry (n = 8). (C) Immunohistochemistry analysis of phospho-p53 at Ser 15. Dark brown color indicates the expression (Scale bar = 20 µm). (D) Comparison of phospho-p53 at Ser15 labeling index. The phospho-p53 at Ser15 positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells. Each bar represents mean±SE (n = 8), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values. (E) Reversal of LETM1-mediated events by MTS-deleted LETM1-adenovirus and shRNA-LETM1. Expression of phospho-Akt1 at Thr308, phospho-Akt1 at Ser473, Akt1, phospho-AMPK at Thr172, AMPK, cyclinD1, phospho-p53 at Ser15 and p53 proteins.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Western Blot, Expressing, Immunohistochemistry, Comparison, Labeling, Staining, Control, shRNA

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: (A) A549 cells that stably expressed mitochondrion-targeted Mito-DsRed were infected with GFP (Ad-GFP), LETM1-adenovirus (Ad-LETM1) or without adenovirus (Control) for 48 h and imaged via confocal microscopy. (B) Mitochondrial ultrastructure was analyzed by transmission electron microscopy in A549 cells. (C) Mitochondrial ultrastructure was analyzed by transmission electron microscopy in the lungs of 9 weeks K -ras LA1 mice. Bottom panels are the magnification of the indicated area (Scale bar = 100 nm).

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Stable Transfection, Infection, Control, Confocal Microscopy, Transmission Assay, Electron Microscopy

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: A549 cells was infected with GFP (Ad-GFP), LETM1 (Ad-LETM1), or without adenovirus (Control). (A) Western blot analysis of the release of cytochrome c from mitochondria. (B) The bands-of-interest were further analyzed by densitometry. Each bar represents mean±SE (n = 3), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values. The 9 weeks K -ras LA1 mice were exposed to aerosols containing adenovirus-LETM1 twice a week for total 4 weeks. Lung tissue homogenates were subjected to Western blot analysis. (C) Expression level of BAX, BclxL, PARP proteins. (D) The bands-of-interest were further analyzed by diameter. (E) Immunohistochemical analysis of cleaved PARP. Dark brown color indicates the expression (Scale bar = 20 µm). (F) Comparison of cleaved PARP labeling index. Cleaved PARP positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 50 cells. Each bar represents mean±SE (n = 8), *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Infection, Control, Western Blot, Expressing, Immunohistochemical staining, Comparison, Labeling, Staining

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: The 9 weeks K -ras LA1 mice were exposed to aerosols containing adenovirus-LETM1 twice a week for total 4 weeks. Lung tissue homogenates were subjected to western blot analysis. (A) Expression of LETM1 and PCNA proteins. (B) The bands-of-interest were further analyzed by densitometry. (C) Immunohistochemistry analysis of LETM1 (upper panel; Scale bar = 20 µm) and PCNA (lower panel; Scale bar = 20 µm). Dark brown color indicates the expression. (D) Comparison of LETM1 (left) and PCNA (right) labeling index. LETM1 and PCNA positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells. Each bar represents mean±SE (n = 8). *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding control values. (E) K -ras LA1 mice lungs with whitish multiple nodules in the lung (green arrows). (F) Histological examination of K -ras LA1 mice lungs (Scale bar = 100 µm).

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Western Blot, Expressing, Immunohistochemistry, Comparison, Labeling, Staining, Control

Journal: PLoS ONE

Article Title: Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

doi: 10.1371/journal.pone.0012535

Figure Lengend Snippet: Summary of tumor incidence in the lungs of K- ras LA1 mice.

Article Snippet: Anti-LETM1 antibody was purchased from Novus Biological (Littleton, CO, USA).

Techniques:

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) LETM1 interactome as determined by affinity purification mass spectrometry (AP-MS). All high confidence interaction partners of LETM1 are shown as nodes. Node color indicates SAINT score, a probability-based measure of interaction confidence. Data are from a single MS experiment. See also (B) Co-immunoprecipitation of MICS1 and LETM1 protein in tandem. Mitochondria were crudely isolated from HEK293 cells and used for immunoprecipitation. The input represents the mitochondrial crude lysate used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP:LETM1) using a LETM1 polyclonal antibody and Protein A/G agarose beads (ProtA/G). ProtA/G beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. Prohibitin was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The right panel illustrates the converse experiment, MICS1 was precipitated (right panel, IP: MICS1) using a MICS1 polyclonal antibody. (C) Native immunoblot of LETM1 (left) and MICS1 (right) show that both proteins can be found in protein complexes of the same size (arrows), MICS1 additionally resides in other protein complexes. (D) Immunoblot analysis of LETM1 and MICS1 expression on conditions of reduced FBS (0.5%) in culture media. Mitochondrial complex III integral subunit UQCRC2 is used as loading control.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Isolation, Co-Immunoprecipitation Assay, Negative Control, Binding Assay, Control, Membrane, Western Blot, Expressing

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) Western blot analysis of LETM1 and MICS1 in HEK293 MICS1WT cell with scramble shRNA (scr) or two different MICS1 knockdowns (KD#1 or KD#2). HSP60 served as a loading control. (B) Proliferation curve of MICS1WT (WT) with a scrambled construct compared to MICS1KD cells (KD) over 4 days using a trypan blue exclusion assay to count cells. Data are means ± SEM (n=3), at 96h statistical analysis using an unpaired student’s t-test (***p<0.001). (C-F) Cellular bioenergetics of MICS1KD cells in various nutrient conditions. Oxygen consumption rate of WT cells with a scrambled control (“WT”) and MICS1KD#1 cells grown in (C) 25 mM glucose, (E) 10 mM galactose for 24 hours before measurement. Data are representative of at least 3 independent experiments. Shown are mean data of triplicate measurements ± SEM. Inhibitors as indicated: A-oligomycin (0.5 μM), B & C-FCCP (0.2 μM each), D-antimycin A/rotenone (0.5 μM). (D & F) Bar charts of XF experiment traces (C & E), data are means of multiple time points after experiment start or drug addition of at least three independent experiments ± SEM. (n=3). Statistical analysis using an unpaired student’s t-test (** p <0.01, *** p <0.001).

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Western Blot, shRNA, Control, Construct, Trypan Blue Exclusion Assay

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: KOAc-induced swelling was measured in mitochondria derived from HEK293 MICS1WT, MICS1KO and MICS1KO cells stably re-expressing MICS1WT (A-B) , HeLa MICS1WT and MICS1KO (C-D) and HeLa LETM1 scramble and LETM1 KD (E-F) cells. MICS1WT: black traces, MICS1KO: red traces, MICS1KO + MICS1WT: blue traces, LETM1scr: black trace, LETM1KD: green trace. (B) Quantification of swelling amplitudes from independent experiments (n=3) HEK293 MICS1WT (black bar, 100 ± 19.71) and HEK293 MICS1KO (red bar, 48.08 ± 11.906). Complementation of MICS1KO with re-expression of MICS1WT restored swelling rates (blue bar, 90.55 ± 12.93). (D) Similar differences in swelling capacities were obtained between HeLa MICS1WT (black bar; 100 ± 9.47) and HeLa MICS1KO (red bar; 63.48 ± 8.60). Lower basal optical density indicates swollen matrix prior KOAc addition; Inhibition of KHE with quinine in HEK293 cells: WT grey bar, 18.14 ± 21.02; KO#1 pink bar, 9.33 ± 28.17; MICS1KO+MICS1, 15.79 ± 10.04. Statistical analysis: One-Way ANOVA with Bonferroni correction (*p <0.05, **p <0.01, ***p <0.001). See also .

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Derivative Assay, Stable Transfection, Expressing, Inhibition

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: Ca 2+ uptake/release dynamics are shown as extramitochondrial Ca 2+ changes of fluorescence intensities of Calcium Green 5N (Ca 2+ 5N) (0.24 μM) (A-L and P-Q) and membrane potential as change of fluorescence intensities of TMRM (330 nM) (M-N) corresponding to the measurement of Ca 2+ fluxes in (K) . Experiments were performed using permeabilized HEK293 MICS1WT, MICS1KO (KO#1, KO#2) ( A-D) and (G-R), MICS1KO#1 + MICS1WT cells was included in (A-B) , and HEK293 LETM1 scr and LETM1KD cells (E-F) in presence of CGP37157 (2 μM). Ca 2+ (10 μM), RR (0.2 μM) and FCCP (2 μM) or alamethicin (2.5 μM) were added when indicated. CsA was added two minutes before measurements in (G-J and Q-R) , nigericin was added 1 min before measurements in absence of thapsigargin or after Ca 2+ uptake in thapsigargin experiments to prevent slowed Ca 2+ uptake dynamics in (I-J) , thapsigargin (1 μM) was added as indicated in K-R , and ADP as indicated in (Q-R) . Quantification of Ca 2+ release rates from independent experiments (n=3) (t: 300-920 s) and statistical analysis: One-Way ANOVA with Bonferroni correction (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001). See also for quantification of Ca 2+ uptake. Quantification of TMRM performed with unpaired two-sided t-test (Welsh correction), *p <0.05. (O-P) CRCs showing that absence of MICS1 supersensitizes mitochondria to Ca 2+ -induced PTP opening by thapsigargin. See also for CRCs. In absence of thapsigargin. Permeabilized HEK293 MICS1WT (O) and MICS1KO#1 (P) cells exposed or not to CsA were subjected to sequential Ca 2+ bolus of 5 µM Ca 2+ and fluorescence intensity was recorded. (Q-R) Thapsigargin-dependent Ca 2+ uptake/release experiments repeated in presence of CsA and ADP in MICS1WT and MICS1KO#1 and KO#2 showing the suppression of Ca 2+ release, quantifications using One-Way ANOVA with Bonferroni correction.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Fluorescence, Membrane

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) Western blot of HEK293 LETM1 scramble (scr) and LETM1KD (KD) used in . PROHIBITIN and β-ACTIN served as mitochondrial and total cellular loading control, respectively. (B) Statistics: unpaired two-sided t-test, *p <0.05, **p <0.01.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Western Blot, Control

Journal: Cell reports

Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

doi: 10.1016/j.celrep.2018.06.002

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-Human LETM1 Polyclonal Antibody , GeneTex , Cat# GTX112455, RRID:AB_1950806.

Techniques: Recombinant, Isolation, Control, Glutathione Assay, Bioassay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: PINK1 interacts with and phosphorylates LETM1. a Expressed PINK1 interacts with LETM1. HEK293 cells were co-transfected with p3xFLAG-CMV-PINK1 and pCMV-3Tag-2a LETM1 (Myc-tag) for 1 day. Myc-LETM1 was IPed from cell lysate with anti-Myc antibody and probed with anti-FLAG antibody by WB. The membrane was reprobed with anti-Myc antibody. b Inversely to a , FLAG-PINK1 protein was IPed with anti-FLAG antibody and probed with anti-Myc and Anti-FLAG antibodies. * is non-special band. c Endogenous PINK1 interacts with LETM1 in human post-mortem brain tissue. Brain lysate was IPed with control IgG or anti-LETM1. The precipitated proteins were probed with anti-PINK1 and reprobed with anti-LETM1 antibodies by WB. d Inversely to c IP was performed with control IgG or anti-PINK1, probed with anti-LETM1 and reprobed with anti-PINK1 antibody by WB. e PINK1 phosphorylates LETM1 at threonine residue(s) in vivo. HEK293 cells were transfected with GFP or pAdtrack-PINK1 for 1 day. LETM1 was IPed with anti-LETM1 antibody, probed with anti-phospho-Thr and reprobed with anti-LETM1 antibody by WB. f PINK1 phosphorylates LETM1 at Thr192 in vitro. Purified bacterial GST-N-LETM1 variants (resides 1–204) were subjected to an in vitro kinase assay with bacterial His-∆N-PINK1 (residues 1–111 deleted) and probed with phospho-Thr antibody. Coomassie blue staining was performed as loading control for GST-N-LETM1. N: nil. g Bacterial His-∆N-LETM1 variants were subjected to in vitro kinase assay with His-∆N-PINK1 and probed for phospho-LETM1 utilizing the Thr192 (pT192) phospho-antibody. Samples were reprobed with anti-LETM1 for loading. h His-∆N-LETM1 was incubated with His-∆N-PINK1 in an in vitro kinase reaction to generate phospho-LETM1. Phospho-LETM1 was then treated without or with CIP for 2 h then probed with pT192 and reprobed with LETM1 antibody. All above experiments were replicated three times, respectively

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Transfection, Membrane, Control, Residue, In Vivo, In Vitro, Purification, Kinase Assay, Staining, Incubation

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: Deficiency or mutant PINK1 reduces phosphorylation of LETM1 at Thr192. a , b Proteins extracted from PINK1 WT or KO MEFs ( a ) or mouse brain ( b ) were subjected to IP with anti-LETM1, probed with pT192 and reprobed with anti-LETM1 by WB. c Proteins extracted from human fibroblast of control (Con) or a PINK1-Q456X patient (Q456X) were subjected to IP with anti-LETM1, probed with pT192 and reprobed with anti-LETM1 by WB. d HEK293 cells were transfected with Adtrack GFP control, AdPINK1-WT, and AdPINK1-Q456X mutant for 1 day. Endogenous LETM1 protein was isolated by IP with anti-LETM1, probed with pT192 and reprobed with anti-LETM1 by WB. e SH-SY5Y cells were treated with 25 µM rotenone for 8 or 16 h. Total cell lysates were either analyzed by WB with anti-PINK1, or subjected to IP with anti-LETM1, probed with pT192 and reprobed with anti-LETM1 antibodies by WB. All above experiments were replicated three times, respectively

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Mutagenesis, Phospho-proteomics, Control, Transfection, Isolation

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: The effect of PINK1-mediated phosphorylation of LETM1 on liposomes calcium release activity in vitro. a About 1 µg purified bacterial full-length His-LETM1-WT, T175E, and T192E were incorporated into liposomes and subjected to calcium release assay. Control (Con) indicates no proteins incorporated. b (top panel) Quantification of calcium release activity measured by rate of calcium release and calculated as change of fluorescence unit (∆ F ). n = 6. (bottom panel) The liposome samples in a were analyzed by WB with anti-LETM1 antibody to show loading of proteins. c HEK293 cells were co-transfected with pAdtrack LETM1 (AdLETM1)-WT or T192A and Adtrack control GFP (AdGFP, WT, T192A) or PINK1 (AdPINK1, pWT, pT192A), and AdLETM1-T192E only for 1 day. The FLAG-LETM1 proteins were isolated by IP with anti-FLAG, probed with pT192 or anti-LETM1 by WB. The experiment was replicated three times. d HEK293 cells were co-transfected with AdLETM1-WT or T192A and AdGFP or AdPINK1, and AdLETM1-T192E only for 1 day. The FLAG-LETM1 proteins were isolated by IP with anti-FLAG and eluted by 3XFLAG peptide. The eluted proteins were subjected to calcium release assay using artificial liposomes. Control is without protein incorporated. e (top panel) Quantification of calcium release activity of d measured by rate of calcium release and calculated as change of fluorescence unit (∆ F ). n = 6. (bottom panel) The liposomes samples in d were subjected to WB with anti-LETM1 antibody to show similar loading of proteins

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Phospho-proteomics, Liposomes, Activity Assay, In Vitro, Purification, Release Assay, Control, Fluorescence, Transfection, Isolation

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: The effect of phospho-LETM1 at Thr192 on mitochondrial calcium transport in primary cortical neurons. a Representative fluorescence traces of mitochondrial Ca 2+ imaging in neurons from PINK1 WT infected with adenovirus GFP, LETM1-WT, T192A, and T192E stimulated by Ac. b Quantification of the rate of calcium transport release activity in a measured by T 1/2 . n = 5. c Representative fluorescence traces of mitochondrial Ca 2+ imaging in neurons from PINK1 KO infected with adenovirus GFP, LETM1-WT, T192A, and T192E stimulated by Ac. d Quantification of the rate of calcium transport release activity in c measured by T 1/2 . n = 5. e Representative traces of mitochondrial Ca 2+ imaging in neurons infected with lenti-4mTG-TN-XXL from PINK1 WT co-infected with lenti-mitoCherry, LETM1-WT, T192A, and T192E stimulated by Ac. f Quantification of the rate of calcium transport in e measured by T 1/2 . n = 4. g Representative traces of mitochondrial Ca 2+ imaging in neurons infected with lenti-4mTG-TN-XXL from PINK1 KO co-infected with lenti-mitoCherry, LETM1-3XFLAG-cherry-WT, T192A, and T192E stimulated by Ac. h Quantification of the rate of calcium transport in g measured by T 1/2 . n = 4

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Fluorescence, Imaging, Infection, Activity Assay

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: The protective role of phospho-LETM1 at Thr192 in mPTP opening and neuronal death. a , b Primary cortical neurons were transfected with control SiCon, SiLETM1-S2, and -S3 and then were processed to calcium-induced mPTP opening assay. a Representative average calcein fluorescence traces of neurons are presented. b Half-life time was calculated for groups in a . n = 5. c Survival assay was performed in siRNA-treated neurons. n = 5. d Cortical neurons from PINK1 WT were infected with lenti-mitoCherry (WC), LETM1-Cherry WT (WW), T192A (WA), or T192E (WE) or from PINK1 KO were also infected with lenti-mitoCherry (KC), LETM1-Cherry WT (KW), T192A (KA), or T192E (KE) for 5 days and then were processed to calcium-induced mPTP opening assay. Representative average calcein fluorescence traces of neurons are presented. n = 5. e Half-life time was calculated for groups in d . n = 5. f Primary cortical neurons from PINK1 WT and KO were infected with adenovirus expression GFP, LETM1-WT, T192A, or T192E and then treated with 75 µM MPP+ or saline for 24 h at DIV3. Neuronal survival was evaluated after MPP+ or saline treatment. n = 5

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Transfection, Control, Fluorescence, Clonogenic Cell Survival Assay, Infection, Expressing, Saline

Journal: Nature Communications

Article Title: PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

doi: 10.1038/s41467-017-01435-1

Figure Lengend Snippet: The protective role of phospho-LETM1 at Thr192 in MPTP-injected mice. a Representative images of the ipsilateral side in PINK1 WT and KO mice injected with corresponding AdGFP, LETM1-WT, T192A, and T192E viruses as indicated with saline or MPTP injected 1 week later. Scale bar: 100 µm. b Quantification of TH-positive dopaminergic neurons from ipsilateral sides in the indicated groups. n = 5. c Quantification of cresyl violet-stained neurons at the ipsilateral sides from the indicated treatment groups. n = 5

Article Snippet: Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT 192 RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).

Techniques: Injection, Saline, Staining