Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Characterization of mitochondrial calpain-5.

doi: 10.1016/j.bbamcr.2021.118989

Figure Lengend Snippet: Fig. 1. Mitochondrial localization and proteolysis/autolysis of calpain-5 in porcine retinas. (a) The purity of the mitochondrial fraction was determined using the appropriate antibodies for the marker proteins [GAPDH, cytosol; AK2, mitochondria; calnexin, ER; LSD1, nucleus]. Highly purified mitochondrial fraction was obtained. (b–e) Antibodies against calpain-5 and specific marker proteins, including beta-actin, Tom20, and PDH, were used; 30 μg of proteins were applied to each lane. Representative results of three independent experiments are shown. (b) Western blot analysis of calpain-5 in proteinase K-treated mitochondria. Isolated mitochondria were treated with 100 μg/mL proteinase K for 30 min at 4 ◦C. Cytosolic fraction and soluble mitochondria in 1% Triton X-100 were used as a positive control to confirm that proteinase K was activated. Calpain-5 was detected in proteinase K-treated mitochondria. (c) Western blot analysis of calpain-5 in Ca2+- incubated mitochondria. Intact forms of cytosolic and mitochondrial calpain-5 were ~75 kDa and ~70 kDa, respectively (lanes 1 and 2). Cytosolic and mitochondrial calpain-5 produced proteolytic/autolytic fragments of ~37 kDa and ~30 kDa, respectively, in the presence of Ca2+ (lanes 3 and 4). (d and e) The effects of calpain inhibitors on proteolysis/autolysis of cytosolic and mitochondrial calpain-5 in the presence of 5 μM PD150606, 10 μM calpeptin, or 1 mM Ca2+ for 120 min at 37 ◦C. Calpeptin inhibited proteolysis/autolysis of calpain-5 in each fraction but PD150606 did not.

Article Snippet: The cells were incubated overnight at 4 ◦C with rabbit polyclonal anti-calpain-5 (1:100, GTX103264, GeneTex) and mouse monoclonal anti-Tom20 antibodies (1:100, sc-136211, Santa Cruz Biotechnology) that were diluted in 1% skimmed milk in PBS-T.

Techniques: Marker, Purification, Western Blot, Isolation, Positive Control, Incubation, Produced

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Characterization of mitochondrial calpain-5.

doi: 10.1016/j.bbamcr.2021.118989

Figure Lengend Snippet: Fig. 3. Immunocytochemistry and western blot analyses of calpain-5 in HeLa cells treated with thapsigargin. (a) Immunocytochemistry of calpain-5, which was mainly localized in the mitochondria. Green, calpain-5; Red, Tom20 (mitochondrial marker); Blue, DAPI. Scale bars: 50 μm. (b) The purity of the mitochondrial fraction was determined using the appropriate antibodies for the marker proteins [GAPDH, cytosol; AK2, mitochondria; calnexin, ER; lamin A/C, nucleus] (20 μg/ lane). Highly purified mitochondrial fraction was obtained. (c) Time-dependency of cytosolic calpain-5 proteolysis/autolysis in thapsigargin-treated HeLa cells. GAPDH was used as a loading control for the cytosolic fraction. Thirty μg of proteins were used in each lane. (d) Time-dependency of mitochondrial calpain-5 proteolysis/autolysis in thapsigargin-treated HeLa cells. Tom20 was used as a loading control for the mitochondrial fraction. Thirty μg of proteins were used for each lane. (e) Western blot analyses of an ER stress marker, ATF6, in thapsigargin-treated HeLa cells. GAPDH was detected as a loading control. Thirty μg of proteins were used in each lane. Representative results of three independent experiments are shown.

Article Snippet: The cells were incubated overnight at 4 ◦C with rabbit polyclonal anti-calpain-5 (1:100, GTX103264, GeneTex) and mouse monoclonal anti-Tom20 antibodies (1:100, sc-136211, Santa Cruz Biotechnology) that were diluted in 1% skimmed milk in PBS-T.

Techniques: Immunocytochemistry, Western Blot, Marker, Purification, Control

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Characterization of mitochondrial calpain-5.

doi: 10.1016/j.bbamcr.2021.118989

Figure Lengend Snippet: Fig. 5. Mitochondrial localization and proteolysis/autolysis of calpain-5 in mouse livers. (a) The purity of the mitochondrial fraction was determined using the appropriate antibodies for the marker proteins [GAPDH, cytosol; AK2, mitochondria; calnexin, ER; LSD1, nucleus]. Highly purified mitochondrial fraction was obtained. (b–f) Antibodies against calpain-5, Tom20 (a marker protein of the mitochondrial outer membrane), and COXIV (a marker protein of the mitochondrial inner membrane) were used; 40 μg of proteins were used for each lane. (b) Western blot analysis of calpain-5 in proteinase K-treated mitochondria. Soluble mitochondria in 1% Triton X-100 were used as a positive control to confirm that proteinase K was activated. Calpain-5 was detected in proteinase K-treated mitochondria. (c) Western blot analysis of cytosolic and mitochondrial calpain-5. Intact forms of mitochondrial and cytosolic calpain-5 were ~ 70 kDa. The truncated form of mitochondrial calpain-5 was ~55 kDa. (d and e) The effects of a calpain inhibitor on the proteolysis/autolysis of cytosolic and mitochondrial calpain-5 in the presence of 10 μM calpeptin or 1 mM Ca2+ for 120 min at 37 ◦C. Calpeptin inhibited proteolysis/autolysis of mitochondrial calpain-5. (f) Time-dependency of mitochondrial calpain-5 proteolysis/autolysis in the presence of 1 mM Ca2+ . The proteolytic/autolytic form of mitochondrial calpain-5 was ~45 kDa. Representative results of three independent experiments are shown.

Article Snippet: The cells were incubated overnight at 4 ◦C with rabbit polyclonal anti-calpain-5 (1:100, GTX103264, GeneTex) and mouse monoclonal anti-Tom20 antibodies (1:100, sc-136211, Santa Cruz Biotechnology) that were diluted in 1% skimmed milk in PBS-T.

Techniques: Marker, Purification, Membrane, Western Blot, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Pyroptotic Macrophage-Derived Microvesicles Accelerate Formation of Neutrophil Extracellular Traps via GSDMD-N-expressing Mitochondrial Transfer during Sepsis

doi: 10.7150/ijbs.87646

Figure Lengend Snippet: Pyroptotic Macrophage-derived MVs Altered Mitochondrial Homeostasis in Neutrophils. Human peripheral neutrophils were isolated and cultured with PBS, control macrophage-derived MVs, or pyroptotic macrophage-derived MVs. (A, B and C) ΔΨ of human peripheral neutrophils was assessed using JC-1staining. JC-1 monomers (green) and aggregates (red) were assessed using fluorescence microscopy (A) and flow cytometry (B and C). Scale bar, 20 µm. (D and E) MitoSOX of human peripheral neutrophils was assessed using fluorescence microscopy (D) and flow cytometry (E). Scale bar, 50 µm. (F and G) Mitochondrial mass in human peripheral neutrophils was detected using flow cytometry (F) and fluorescence microscopy (G). Scale bar, 20 µm. (H) Representative images showing neutrophil staining of DNA (DAPI, blue), TOM20 (green), and 8-OHG (red) in human peripheral neutrophils following exposure to MVs for 4 h. Results are represented as mean ± SEM.

Article Snippet: Cells were blocked with 1% BSA for 30 min and stained with rabbit anti-histone H3 antibody (Abcam; 1:500); mouse anti-myeloperoxidase antibody (Abcam 1:500); rabbit anti-GSDMD (Abcam; 1:500); rabbit anti-TOM20 antibody (Abcam 1:500); mouse anti-8-OHdG antibody (Novus; 1:200).

Techniques: Derivative Assay, Isolation, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Biallelic NSUN3 Variants Cause Diverse Phenotypic Spectrum Disease: From Isolated Optic Atrophy to Severe Early-Onset Mitochondrial Disorder

doi: 10.1167/iovs.66.6.17

Figure Lengend Snippet: Affected individuals from Family 1, Family 2 and Family 3 displayed COX-negative fibers, diminished OXPHOS activity, and reduced amounts of some OXPHOS components in skeletal muscle. Skeletal muscle biopsy from affected individual III-2 of family 3 (A–C) . (A) H&E staining showing mild myopathic changes with rounding of muscle fibers, variation in fiber size and internal nuclei (arrowhead). (B) Cytochrome c oxidase (COX) staining displays fibers with markedly reduced COX activity ( dashed lines ). (C) Succinate dehydrogenase (SDH) staining present fibers with subsarcolemmal positive deposits ( arrows ). (D–F) Oxidative phosphorylation (OXPHOS) complex activity in affected individuals II-1 of family 1 (D) , II-1 of family 2 (E) , and III-2 of family 3 (F) . Patient muscle homogenate ( red line ) showing reduced complex IV activity for all three subjects and diminished complex I activity for patient II-2 of family 1. Gray bars represent the normal range. (G) Representative example of WB analysis of muscle homogenates for ATP5A, UQCRC2, SDHB, COX II, NDUFB8, TOM20 and β-ACTIN of healthy controls (C1, C2), and affected individuals III-2 and II-1 from family 3. (H) Quantification of 5 WB experiments of probands III-2 and II-1 from family 3 for ATP5A, UQCRC2, SDHB, COX II, NDUFB8 and TOM20 normalized to β-ACTIN. Data represent the mean value ± SD and were statistically analyzed by one-way ANOVA followed by a Sidak test as a post hoc test for multiple comparisons. (I–L) Representative electron micrographs depicting mitochondrial positioning in skeletal muscle of affected individual III-2. Many mitochondria were swollen and elongated, stretching the length of a sarcomere ( arrows , I–J ). In some fibers mitochondria were devoid of cristae ( asterisk , K ) and had linearized cristae membranes ( arrowhead , L ). Scale bars : 50 µm (A–C) , 0.5 µm (I–L) .

Article Snippet: The following primary antibodies (Abs) were used for immunocytochemistry (ICC,) Western blotting (WB), or both: mouse monoclonal antibodies (mAbs) to cytochrome c oxidase subunit I (Cox I; ICC: 1:100; Molecular Probes, Eugene, OR, USA), OxPhos Human WB Antibody Cocktail (WB: 1:1000; Invitrogen, Carlsbad, CA, USA), and β-Actin (WB: 1:1000; Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit mAb to Tom20 (ICC: 1:200, WB: 1:1000; Cell Signaling Technology, Danvers, MA, USA), and chicken polyclonal Ab to Vimentin (WB: 1:10000; Novus Biologicals, Littleton, CO, USA).

Techniques: Activity Assay, Staining, Phospho-proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: Biallelic NSUN3 Variants Cause Diverse Phenotypic Spectrum Disease: From Isolated Optic Atrophy to Severe Early-Onset Mitochondrial Disorder

doi: 10.1167/iovs.66.6.17

Figure Lengend Snippet: Strongly reduced COX I signals, reduced OXPHOS complex IV and I protein amounts and reduced OXPHOS activity in dermal fibroblasts of affected individuals of family 3. (A) Immunocytochemistry staining (ICC) of human dermal fibroblasts of a healthy control and affected individuals III-2 and II-1 of family 3. Anti-COX I (Alexa Flour 647), anti-TOM20 (AF 555), Phalloidin-Atto 488 and nuclei (DAPI). Patient fibroblasts exhibit markedly reduced COX I staining intensities. Scale bars : 10 µm. (B) Representative example of WB analysis of ATP5A, UQCRC2, SDHB, COX II, NDUFB8, TOM20 and VIMENTIN of healthy controls (C1–C3), affected individuals III-2 and II-1 of dermal fibroblast protein lysates. (C) Quantification of 6 WB experiments for ATP5A, UQCRC2, SDHB, COX II, NDUFB8 and TOM20 normalized to VIMENTIN. (D) OCR of dermal fibroblasts of healthy controls (C4, C5) and NSUN3 patients measured with the Seahorse Cell Mito Stress Assay. OCR was recorded during sequential injections of drug inhibitors: oligomycin; FCCP; antimycin A and rotenone. Quantification of the OCR at basal respiration levels, ATP production levels and maximal respiration; cells of affected individuals II-1 and III-2 show a significant reduction of respiratory capacity and ATP production. Data represent the mean value ± SD and were statistically analyzed by one-way ANOVA followed by a Sidak test as a post hoc test for multiple comparisons.

Article Snippet: The following primary antibodies (Abs) were used for immunocytochemistry (ICC,) Western blotting (WB), or both: mouse monoclonal antibodies (mAbs) to cytochrome c oxidase subunit I (Cox I; ICC: 1:100; Molecular Probes, Eugene, OR, USA), OxPhos Human WB Antibody Cocktail (WB: 1:1000; Invitrogen, Carlsbad, CA, USA), and β-Actin (WB: 1:1000; Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit mAb to Tom20 (ICC: 1:200, WB: 1:1000; Cell Signaling Technology, Danvers, MA, USA), and chicken polyclonal Ab to Vimentin (WB: 1:10000; Novus Biologicals, Littleton, CO, USA).

Techniques: Activity Assay, Immunocytochemistry, Staining, Control