66562 1 ig  (Proteintech)

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: Functionalization of an AFM microcantilever with a single mitochondrion. Isolated mito-GFP is collected by a anti-TOMM22 antibody precoated microcantilever. Scale bar, 10 mm.

Article Snippet: Next, the cantilevers were coated with anti-TOMM22 antibody-conjugated microbeads (Miltenyi Biotech, 130-127-693) for 1 h at 25 °C.

Techniques:

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: a. Schematic of AFM microcantilever functionalization. A silicon microbead (5 µm diameter), which had been glued to the free end of the microcantilever, is pre-coated with concanavalin A. Afterwards anti-TOMM22 antibody-conjugated nanobeads (50 nm, diameter) are attached to the microbead. b. Schematic of an isolated mitochondrion attached to the functionalized microcantilever bead. Anti-TOMM22 antibodies specifically bind TOMM22 proteins at the outer membrane of mitochondria. c. Attaching an isolated mito-GFP to the microbead glued to the microcantilever. White arrows, mito-GFP. d. A microcantilever functionalized with a mito-GFP (green). e. Schematic of an experiment for mitochondria-cell adhesion profiling with AFM-based single-cell force spectroscopy. The mitochondria-attached AFM microcantilever is approached to the cell surface with 2 µm s −1 (1), resulting in mitochondria-cell contact (~100 ms) (2), followed by a retraction with 2 µm s −1 (3), finalizing in mitochondria-cell separation (4). f. Schematic diagram of a construct designed to display the anti-GFP nanobody on the cell surface together with cytosolic mCherry (red) expression (top). Brightfield and fluorescence images of a HEK293T cell expressing the cell-surface anti-GFP nanobody and cytosolic mCherry (bottom). Black and white arrows indicate cells not expressing the anti-GFP nanobody and mCherry. Red arrows indicate a cell positive for the anti-GFP nanobody and mCherry. White dashed lines highlight the AFM microcantilever. The construct was validated in at least three independent experiments. g. Schematic of mitochondria-cell adhesion profiling in HEK293T cells expressing the cell surface anti-GFP nanobody and cytosolic mCherry with mitochondria displaying GFP. h. Example force-distance retraction curves. Total adhesion forces are indicated with dark red arrows. Untreated cells were used as a control. i. Quantification of mitochondrion-cell adhesion force per individual approach. Control: n = 112 (total 22 cells); anti-GFP nanobody: n = 95 (total 29 cells), P < 0.0001, two-sided Mann-Whitney U test. j. Distribution of rupture events per individual approach. k. Quantification of single mitochondrion-cell adhesion force averaged per cell. Control: n = 22, anti-GFP nanobody: n = 29, P < 0.0001, two-sided Mann-Whitney test. l. Quantification of single mitochondrion-cell adhesion force averaged per cell. Control: n = 30, anti-mCD71 nanobody: n = 29, P < 0.0001, two-sided Mann-Whitney test. m. Schematic of the mitochondrion-cell adhesion profiling in the presence of heparan sulfate. Isolated mito-GFP were pre-incubated with heparan sulfate before measuring their adhesion force to either control cells or anti-GFP nanobody expressing cells. n. Force-distance retraction curves (average from n > 25 selected curves per condition). Control cells (top) and cells expressing cell surface anti-GFP nanobody (bottom) were used to profile the adhesion to mito-GFP pre-coated with heparan sulfate. The anti-GFP nanobody-positive cells were identified via cytoplasmic mCherry fluorescence. o. Quantification of mitochondrion-cell adhesion force per approach. Nine data points for the anti-GFP nanobody + heparan sulfate (HS) condition are not shown for plotting. Control: n = 112; Control + HS: n = 110; anti-GFP nanobody + HS: n = 83; Control vs. Control + HS: P < 0.0002, anti-GFP nanobody + HS vs. Control + HS: P < 0.0001, anti-GFP nanobody + HS vs. Control: P < 0.0001, two-sided Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. p. Quantification of mitochondrion-cell adhesion force averaged per cell. One data point for the anti-GFP nanobody + HS condition is not shown for plotting. Control: n = 22; Control + HS: n = 28; anti-GFP nanobody + HS: n = 27; Control vs. Control + HS: P < 0.5172, anti-GFP nanobody + HS vs. Control + HS: P < 0.0001, anti-GFP nanobody + HS vs. Control: P = 0.0006, Kruskal-Wallis test corrected with Dunn’s test for multiple comparisons. q. Schematic (top) and quantification (bottom) of the efficacy of the delivery of nanobody-displaying mitochondria pre-coated with heparan sulfate into CD71-positive T cells four hours after transplantation. n = 6, Control nanobody vs. Control nanobody + HS: P = 0.0152, anti-CD71 nanobody vs. anti-CD71 nanobody + HS: P = 0.0931, two-sided Mann-Whitney test. See Supplementary Fig. for flow cytometry graphs. * P < 0.05, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m for q and median for i, k, l, o, p. Scale bars, 5 µm (c, d) and 10 µm (f).

Article Snippet: Next, the cantilevers were coated with anti-TOMM22 antibody-conjugated microbeads (Miltenyi Biotech, 130-127-693) for 1 h at 25 °C.

Techniques: Isolation, Membrane, Single Cell, Force Spectroscopy, Construct, Expressing, Fluorescence, Control, MANN-WHITNEY, Incubation, Transplantation Assay, Flow Cytometry

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Volcano plot shows interactors of PINK1-YFP (red) identified by AP-MS following transduction with CTRL or PINK1 guides in HeLa PINK1-YFP cells. Cells in both groups were treated with 10 µM CCCP overnight. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. ( B ) Flow cytometry measurements of PINK1-YFP intensity (top) and intensity of MTS-mCh (bottom) from the same HeLa PINK1-YFP+MTS-mCh cells. N = 6 biological replicates measured from two independent transductions. All statistical comparisons are to the vehicle-treated CTRL guide group. Exact P values, ns P = 0.2179, **** P ≤ 0.0001 (exact P values P = 1.2e-14 for all comparisons) by two-way ANOVA with Dunnett’s multiple comparison test. Error bars mean +/− SD. ( C ) Representative confocal Airyscan images shown as z-projections in HeLa PINK1-YFP cells immunostained for TOMM70 to mark mitochondria. The arrow indicates PINK1-YFP accumulated around lipid droplets; the arrowhead indicates PINK1 co-localizing with TOMM70. Scale bar = 10 µm. ( D ) CLEM images demonstrating that the PINK1-YFP accumulates around lipid droplets (LD) following loss of the TOM translocase (HeLa PINK1-YFP cells). ER (yellow arrowheads) and small vesicles (red arrows) were adjacent to lipid droplet collections and accumulated PINK1-YFP. Scale bars: green = 20 µm; yellow = 5 µm; black = 1 µm; white = 500 nm. ( E ) Representative confocal images of TIMM23 KO pools in HeLa PINK1-YFP cells showing PINK1-YFP accumulates on mitochondria in cells lacking TIMM23 by immunostaining. Scale bar = 10 µm. ( F ) Line scan of dotted line in ( E ). ( G ) Quantification of ( E ). **** P ≤ 0.0001 (exact P value P = 9.0037e-05) via two-tailed unpaired t test with Welch’s correction. N = 4 wells analyzed, plated on two separate occasions from the same KO pool. Cells were analyzed 7 or 8 days after electroporation, and cells without TIMM23 were scored for PINK1-YFP accumulated on TOMM40-positive mitochondria. Error bars mean +/− SD. ( H ) Representative immunoblots from HeLa cells with exogenous PINK1-YFP (left blot) or endogenous PINK1 (right blot) +/− 10 µM CCCP treatment for 4 h, probing for PINK1 activation and stabilization. N ≥ 3 independent experiments. ( I ) Volcano plot shows PINK1-YFP (red) interactors, following CTRL vs. TIMM23 KD in HeLa PINK1-YFP cells. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. ( J ) In gel fluorescence of PINK1-YFP complexes separated by CN-PAGE after mixing lysates with the indicated antibodies. The specific interaction of the antibody with PINK1-YFP-containing complex increases the molecular weight of the complex, causing it to shift up in the gel. 10 µM CCCP treatment in the indicated lanes was for 3 h. N = 2 independent experiments with TOMM20 gel shift, one of which also tested TOMM22 and TOMM40 antibodies. (HeLa PINK-YFP cells). .

Article Snippet: TOMM22 (Proteintech, 66562-1-Ig), TOMM40 (Proteintech, 18409-1-AP), TOMM20 (Santa Cruz, sc-17764), and CHCHD2 (Proteintech, 66302-1-Ig).

Techniques: Protein-Protein interactions, Transduction, Flow Cytometry, Comparison, Immunostaining, Two Tailed Test, Electroporation, Western Blot, Activation Assay, Fluorescence, Clear Native PAGE, Molecular Weight, Gel Shift

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Representative confocal images of TOMM22, TOMM40, TIMM23 KO pools in HeLa PINK1-YFP+MTS-mCh cells showing PINK1-YFP accumulates in the same pattern as observed by CRISPRi. Images were obtained 7 or 8 days after electroporation with Cas9 ribonucleoprotein complexes. Scale bar 10 = µm. ( B ) Total protein measured via SimplyBlue SafeStain of same gel as in (Fig. ), demonstrating equal loading. ( C ) Flow cytometry data of HeLa MFN2-Halo cells + BFP-Parkin transfected with ATP5MG-mCherry-sfGFP and treated +/− 10 µM CCCP for 4 h, illustrating differences in MFN2-Halo levels in the presence of the clogger. ns P = 0.5976, **** P ≤ 0.0001 (exact P value P < 1e-15) by two-way ANOVA with Tukey’s multiple comparisons test. Error bars mean +/− SD. N = 3 independent experiments, 9 replicates. ( D ) Flow cytometry data of HeLa MFN2-Halo cells transfected with ATP5MG-mCherry-sfGFP and treated +/− 10 µM CCCP for 4 h, illustrating differences in MFN2-Halo levels in the presence of the clogger. ** P = 0.0086, **** P ≤ 0.0001 (exact P value P = 7.4e-09) by two-way ANOVA with Tukey’s multiple comparisons test. Error bars mean +/− SD. N = 3 independent experiments, 10 replicates. ( E ) Flow cytometry data of HeLa PINK1-YFP cells expressing TOMM70 endogenously tagged with HaloTag and IMMT-DHFR clogger. Cells were treated +/− 20 µM CCCP for 4 h, demonstrating differences in PINK1-YFP stabilization. ns P = 0.4962, *** P = 0.0004 by two-way ANOVA with Šídák’s multiple comparisons test. Error bars mean +/− SD. N = 6 replicates run on two different occasions (separate occasions denoted by open or closed circles). ( F ) Flow cytometry of HeLa PINK1-YFP cells. *** P = 0.0003, **** P ≤ 0.0001 (exact P values - CTRL vs PAM16, P = 1.1e-05; CTRL vs TIMM23, P = 9.3e-07; CTRL vs NDUFAB1, P = 9e-06; CTRL vs PMPCB, P = 3.6e-07; TIMM44 vs TIMM23, P = 1.2e-06; PAM16 vs TIMM23, 1.3e-08). Error bars mean +/− SD. N = 6 replicates (5 for TIMM23) from 2 independent transductions. ( G ) Representative 2D kernel density plots comparing single-cell PINK1-YFP intensity and intensity of the MMP sensitive dye MitoLite NIR as in (Fig. ).

Article Snippet: TOMM22 (Proteintech, 66562-1-Ig), TOMM40 (Proteintech, 18409-1-AP), TOMM20 (Santa Cruz, sc-17764), and CHCHD2 (Proteintech, 66302-1-Ig).

Techniques: Electroporation, Flow Cytometry, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: Simvastatin downregulates TOMM40 and TOMM22 in both C2C12 and hSkMC skeletal muscle. Differentiated C2C12 and primary hSkMC myotubes were treated with 2 µM simvastatin for 24 h. ( A ) A schematic diagram of the mammalian TOM complex, consisting of seven subunits, located in the outer mitochondrial membrane. ( B ) Simvastatin treatment (2 µM) downregulates Tomm40 , Tomm22 , Tomm20 , and Tomm5 in differentiated C2C12 myotubes. ( C ) Simvastatin treatment (2 µM) downregulates TOMM40 and TOMM22 in hSkMC myotubes. Numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01 vs. 0 µM simvastatin by the Student’s t -test ( n = 3 biological replicates).

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Membrane

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: TOMM40 and TOMM22 knockdown decreases the mitochondrial oxygen consumption rate and promotes mitochondrial superoxide production. ( A ) Schematic illustration of the two-step transfection and differentiation experiment in C2C12 cells in vitro. ( B ) Confirmation of Tomm40 (T40) and Tomm22 (T22) KD in C2C12 myotubes by ~92% and ~85%, respectively, measured with qPCR ( n = 3 biological replicates). ( C ) Oxygen consumption rates of C2C12 myotubes transfected with Tomm40 and Tomm22 siRNAs vs. NTC were quantified using the Seahorse 96e Extracellular Flux Analyzer. With the addition of oligomycin, FCCP, and Antimycin A + Rotenone, basal respiration, ATP production, and maximal respiration were quantified. ( D – F ) The same experiment carried out in C2C12 cells ( C , D ) was conducted in primary hSkMCs. ( G ) Mitochondrial superoxide production with Tomm40 and Tomm22 KD in C2C12 cells was quantified by MitoSOX fluorescence probe. All values were normalized to protein concentration by the Bradford assay. All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. NTC. One-way ANOVA, with Tukey’s post hoc test to identify differences between groups (unless specified, n = 4–6 biological replicates).

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Knockdown, Transfection, In Vitro, Fluorescence, Protein Concentration, Bradford Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: Tomm40 and Tomm22 regulate mitochondrial cholesterol content and CoQ levels in C2C12 myotubes. ( A ) Subcellular fractionation was performed to isolate crude mitochondria from whole cells. ( B ) Total and free cholesterol levels in mitochondria isolated from C2C12 myotubes transfected with NTC, Tomm40 , and Tomm22 siRNAs, singly and in combination. ( C , D ) In a separate experiment, after cells were transfected with siRNAs, 50 µg/mL LDL-C was added to cell media for 24 h. Total and free cholesterol were quantified in the mitochondria using the Amplex Red Cholesterol Assay. (“Ctrl” = control group, “+ LDL-C” = LDL-C treatment group) ( E ) OCR levels were assessed in C2C12 cells after the LDL-C addback, and ( F ) basal respiration and ATP production were quantified using Wave Desktop 2.6 software ( n = 10–12 biological replicates). ( G ) Total CoQ from isolated mitochondria of NTC, Tomm40 , Tomm22 , and Tomm22/40 KD C2C12 cells were quantified by LC-MS/MS. All values were normalized to protein concentration, measured by BCA. All graphical and numeric data represent the mean ± SEM * p < 0.05, ** p < 0.01 vs. NTC (without LDL-C addback) by the Welch and Brown–Forsythe ANOVA or one-way ANOVA, with Tukey’s post hoc test to identify differences between groups (unless specified, n = 3–6 biological replicates).

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Fractionation, Isolation, Transfection, Amplex Red Cholesterol Assay, Control, Software, Liquid Chromatography with Mass Spectroscopy, Protein Concentration

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: TOMM40 and TOMM22 knockdown, singly and in combination, impairs mitochondrial dynamics in skeletal myotubes. ( A ) TEM micrographs of NTC, Tomm40, Tomm22, and Tomm22/40 KD in C2C12 cells. Arrowheads indicate mitochondrial fission events. Analysis of mitochondrial morphology using ImageJ software: ( B ) average mitochondrial length (nm) and ( C ) average mitochondrial width (nm). ( n = 10–15 cells). ( D ) Mitochondrial fission ( Fis1/FIS1 , Drp1/DNM1L ) and fusion ( Mfn2/MFN2 , Opa1/OPA1 ) markers were quantified by qPCR in NTC, Tomm22, Tomm40, and Tomm22/40 KD C2C12 cells. ( E ) The experiment was conducted as in ( D ) but with hSkMCs ( n = 3 biological replicates). ( F ) Mitochondrial density was quantified using a MitoTracker™ Deep Red FM fluorescence probe to measure the average fluorescence intensity and normalized to protein concentration by the Bradford assay ( n = 10–12 biological replicates). ( G ) Relative mtDNA copy number levels were quantified and normalized to the B2m transcript levels (nuclear DNA) by qPCR in siRNA transfected C2C12 cells ( n = 3 biological replicates). All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. NTC. One-way ANOVA, with Tukey’s post hoc test to identify differences between groups.

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Knockdown, Software, Fluorescence, Protein Concentration, Bradford Assay, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: TOMM40 and TOMM22 knockdown, singly and in combination, promotes mitochondrial damage and mitophagy in skeletal muscle in vitro and in vivo. ( A ) TEM micrographs of C2C12 myotubes representing types 1 (healthy; A1 image of NTC), 2 (unhealthy; A2 image of Tomm40 KD), and 3 (damaged/ruptured; A3 image of Tomm22/40 KD) mitochondria (scale bars = 1 μm; inset scale bars = 200 nm). ( B ) Analysis of mitochondrial morphology and damage in NTC vs. KD C2C12 cells. Bar graph represents percent of cells exhibiting type 1 mitochondria. ( C ) Bar graph represents the percent of types 2 and 3 mitochondria in NTC vs. KD C2C12 cells (for 5B, C, and D: n = 10–15 cells, number of mitochondria assessed per cell > 200). ( D ) Percent of mitophagosomes per total number of mitochondria per cell, identified from the TEM images ( n = 10–15 cells). ( E ) mRNA transcript levels of PINK1 and PRKN (mitophagy) were quantified using qPCR in NTC vs. KD hSkMCs ( n = 3 biological replicates). ( F ) Representative Western blot of PRKN protein expression in hSkMCs compared with GAPDH control. ( G ) mRNA transcript levels of Tomm40 (~50% knockdown) were quantified in the gastrocnemius skeletal muscle of male mice injected with AAV8 scrambled vs. Tomm40 shRNA ( n = 6/group). ( H ) Representative TEM images of gastrocnemius muscle samples from scrambled vs. Tomm40 shRNA ( n = 6/group) male mice. ( I ) Bar graph represents the average number of mitochondria within a surface area of 36 µm 2 , ( J ) length of intermyofibrillar mitochondria (as seen in the TEM images), ( K ) length of sarcolemnal mitochondria, and ( L ) percent of ruptured mitochondria per 36 µm 2 . All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. NTC or scrambled shRNA by one-way ANOVA, with Tukey’s post hoc test to identify differences between groups.

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Expressing, Control, Injection, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: TOMM40 and BCAP31 overexpression rescues mitochondrial respiration, ATP production, and proton leak after simvastatin treatment of skeletal muscle cells. ( A ) OCR was measured in C2C12 cells treated with 2 µM simvastatin + empty vector or Tomm22 , Tomm40 , Tomm20 ( T20 ), or Tomm20/22/40 expressing plasmids for 24–48 h. Basal respiration and ATP production determined from the OCR analysis. ( B ) The same experiment was conducted in hSkMCs treated with 2 µM simvastatin + empty vector or TOMM22 , TOMM40 , TOMM22/40 expressing plasmids for 24–48 h ( n = 10–15 biological replicate). ( C ) mitoROS was determined in C2C12 cells by quantifying the mean mitoSOX™ fluorescence intensity and normalizing to the total protein concentration using the Bradford assay ( n = 10–12 biological replicates). mRNA transcripts of ( D ) Bcap31 in C2C12 and ( E ) BCAP31 in hSkMC myotubes without and with 2 µM simvastatin was quantified by qPCR. ( F – I ) Basal respiration, ATP production, maximal respiration, and proton leak were analyzed from OCR recordings in C2C12 cells treated with 2 µM simvastatin + empty vector or Bcap31 , Bcap31/Tomm22 , or Bcap31/Tomm40 expressing plasmids for 24–48 h. ( J – M ) Basal respiration, ATP production, maximal respiration, and proton leak were analyzed from OCR recordings in hSkMCs treated with 2 µM simvastatin + empty vector or BCAP31 , BCAP31/TOMM22 , or BCAP31/TOMM40 expressing plasmids for 24–48 h. ( N ) Total and free cholesterol were quantified in the mitochondria of C2C12 myotubes using the Amplex Red Cholesterol Assay ( n = 3–6 biological replicates). ( O ) Total CoQ 9 was quantified from mitochondria isolated from C2C12 cells transfected with empty vector (control), 2 µM simvastatin + empty vector, Tomm22 , Tomm40 , Tomm20 , or Tomm20/22/40 expressing plasmids ( n = 3 biological replicates). All values were normalized to protein concentration by the BCA. All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. EV by Welch and Brown–Forsythe or one-way ANOVA, with Tukey’s post hoc test to identify differences between groups.

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Over Expression, Plasmid Preparation, Expressing, Fluorescence, Protein Concentration, Bradford Assay, Amplex Red Cholesterol Assay, Isolation, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: Overexpressing TOMM40 and TOMM22 , singly and in combination, suppresses statin-induced mitochondrial fission and promotes fusion in skeletal muscle cells. ( A ) Representative TEM images of mitochondrial morphology in C2C12 cells transfected with empty vector (EV), 2 µM simvastatin + EV, and Tomm22 , Tomm40 , or Tomm22/40 expressing plasmids. ( B , C ) Using ImageJ software analysis in conjunction with the TEM images in ( A ), the average mitochondrial length and width (nm) were measured ( n = 10–15 cells). ( D ) Mitochondrial fission ( Fis1/FIS1 , Drp1/DNM1L ) and fusion ( Mfn2/MFN2 , Opa1/OPA1 ) markers were quantified by qPCR in EV and 2 µM simvastatin + EV, Tomm22 , Tomm40 , Tomm20 , and Tomm20/22/40 ( T20/22/40 ) overexpressing C2C12 cells. ( E ) The same experiment was conducted as in ( D ) but with hSkMCs ( n = 3 biological replicates). ( F ) Mitochondrial density was quantified using a MitoTracker™ Deep Red FM fluorescence probe to measure the average fluorescence intensity and was normalized to protein concentration by the Bradford assay ( n = 10–12 biological replicates). ( G ) Relative mtDNA copy number levels were quantified and normalized to the B2m transcript levels (nuclear DNA) by qPCR in siRNA transfected C2C12 cells ( n = 3 biological replicates). All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA, with Tukey’s post hoc test to identify differences between groups, was performed.

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Transfection, Plasmid Preparation, Expressing, Software, Fluorescence, Protein Concentration, Bradford Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting TOMM40 and TOMM22 to Rescue Statin-Impaired Mitochondrial Function, Dynamics, and Mitophagy in Skeletal Myotubes

doi: 10.3390/ijms262210977

Figure Lengend Snippet: Overexpression of TOMM40 and TOMM22 , but not TOMM20 , rescues simvastatin-induced mitophagy leading to reduced mitochondrial damage. ( A ) TEM micrographs of mitophagosomes found in EV, 2 µM simvastatin + EV, and 2 µM simvastatin + Tomm22/Tomm40 overexpressing C2C12 cells. Arrowheads indicate mitophagosomes. ( B ) Analysis of mitochondrial morphology and damage in EV or 2 µM simvastatin + EV, T22 , T40 , or T22/40 overexpression in C2C12 cells. Bar graph represents percent of cells exhibiting type 1 mitochondria. ( C ) Bar graph represents percent of types 2 and 3 mitochondria in EV or 2 µM simvastatin + EV, T22 , T40 , or T22/40 overexpression in C2C12 cells ( n = 10–15 cells). ( D ) Percent of mitophagosomes per total number of mitochondria per cell, identified from TEM images ( n = 10–15 cells). ( E , F ) mRNA transcript levels of Pink1 / PINK1 and Prkn/PRKN (mitophagy) were quantified using qPCR in EV vs. 2 µM simvastatin + EV, TOMM22 , TOMM40 , TOMM20 , or TOMM20/22/40 in ( E ) C2C12 and ( F ) hSkMC myotubes ( n = 3 biological replicates). All graphical and numeric data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. EV by one-way ANOVA, with Tukey’s post hoc test to identify differences between groups. p < 0.05 for a vs. b vs. c by two-way ANOVA, with Sidak’s multiple comparisons test.

Article Snippet: Membranes were then incubated with primary antibodies: TOMM40 (E6Q3Z), TOMM22 (E3F4M), TOMM20 (D8T4N), VDAC1 (D73D12), MFN2 (D1E9), OPA1 (D6U6N), DRP1 (D6C7), GAPDH (14C10) rabbit monoclonal antibodies, and PARKIN (Prk8) mouse monoclonal antibody (Cell Signal) diluted 1:1000 ( v / v ) in TBST overnight on a rotating platform at 4 °C.

Techniques: Over Expression