Journal: Frontiers in Physiology

Article Title: Mesenchymal Stem Cells Shift Mitochondrial Dynamics and Enhance Oxidative Phosphorylation in Recipient Cells

doi: 10.3389/fphys.2018.01572

Figure Lengend Snippet: Mitochondrial morphology and mtDNA of fixed fibroblasts and live fibroblasts co-cultured with MSCs. (A) Representative images demonstrating assessment of mitochondrial morphology and mtDNA of fixed fibroblasts from controls and patients with clinically diagnosed mitochondrial disease. Images were collected using immunofluorescence against TOMM20 (mitochondria) and DNA (DNA). (B) Representative images demonstrating assessment of mitochondrial morphology and mtDNA of live fibroblasts (from controls and patients with a clinically diagnosed mitochondrial disease) co-cultured with MSCs. Images were collected following fluorescent labeling of fibroblasts with MitoTracker Deep Red (mitochondria) and PicoGreen (DNA). MSCs were separately fluorescently labeled with MitoTracker Red (mitochondria) prior to co-culture. Images from both panels were collected using confocal microscopy.

Article Snippet: Cells were permeabilized with 0.5 mL 0.2% Triton X-100 (Sigma; Oakville, ON) in PBS for 15 min, washed with PBS, and then blocked with 10% FBS (Life Technologies) in PBS for 30 min. Cover slips were simultaneously incubated with primary antibodies for the outer mitochondrial membrane protein TOMM20 (FL-145, Santa Cruz; Dallas, TX) and DNA (CBL-186, EMD Millipore; Etobicoke, ON) diluted to 1:1,000 in 5% FBS, 95% PBS (Life Technologies) at 37°C for 1 h. Cells were washed 3 x with PBS and then incubated with secondary antibodies (1:5,000) at RT for 1 h. The following secondary antibodies conjugated to fluorescent dyes were used: Alexa Fluor 488 goat anti-mouse IgG (TOMM20, Molecular Probes, Eugene, OR) and Alexa Fluor 647 goat anti-rabbit IgG (DNA, Molecular Probes).

Techniques: Cell Culture, Immunofluorescence, Labeling, Co-Culture Assay, Confocal Microscopy

Journal: Disease Models & Mechanisms

Article Title: Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

doi: 10.1242/dmm.047134

Figure Lengend Snippet: Mitochondrial morphology is altered by ammonia. Human astrocytoma cells were transfected with pEGFP-Mito to visualize mitochondria. In primary rat astrocytes, visualization was achieved by immunostaining against Tom20. Cells were treated with 5 mM NH 4 Cl for respective durations. At least 20 pictures were taken per sample, showing approximately three to five cells each. Mitochondria were categorized as tubular, intermediate and fragmented morphological phenotype. (A) Representative images of morphological phenotype characterization in human astrocytoma cells. (B) Changes in morphology of mitochondria after treatment with 5 mM NH 4 Cl in primary rat astrocytes (top row) and human astrocytoma (bottom row). (C,D) Time course of changes in mitochondrial morphology in human astrocytoma ( n =3-6) (C) and primary rat astrocytes ( n =3) (D). Data are presented as mean±s.e.m. Statistics: one-way ANOVA with Dunnett's post test (all treatments versus control) for tubular morphology. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: To determine mitochondrial morphology changes in primary rat astrocytes, mitochondria were visualized by immunostaining against Tom20 (primary antibody: sc-11415, Santa Cruz Biotechnology, Dallas, TX, USA; secondary antibody: Alexa Fluor 488, #A27034, Thermo Fisher Scientific).

Techniques: Transfection, Immunostaining, Control

Journal: Redox biology

Article Title: ROS-mediated cytoplasmic localization of CARM1 induces mitochondrial fission through DRP1 methylation.

doi: 10.1016/j.redox.2024.103212

Figure Lengend Snippet: Fig. 1. CARM1 regulates mitochondrial dynamics and function. (A,B) Gene Set Enrichment Analysis results from RNA-sequencing of public data (GSE152666). Enrichment plot generated upon Kyoto Encyclopedia of Genes and Genomes database analysis (A) and heatmap of the core enriched genes of the gene set oxidative phosphorylation pathway (B). (C) OCR upon injection of Oligo, CCCP, and R/A into CARM1-WT or -KO MEF cells. Error bars represent mean ± SD (n = 3). *p < 0.05 and **p < 0.01. (D) Confocal images of MitoTracker™ Red (red), CARM1 (green), and DAPI (blue) in CARM1-WT or -KO MEF cells. The intensity of MitoTracker™ Red indicates the mitochondrial membrane potential. Scale bars, 10 μm. (E) Confocal images of TOM20 (red) in CARM1-WT or -KO MEF cells. Scale bars, 10 μm. (F) Mitochondrial morphology was analyzed from the confocal images using ImageJ/FiJi software. The more elongated the mitochondria, the higher the mitochondrial interconnectivity. Data are presented as mean ± SD (n = 5). ***p < 0.001. (G) Western blots of CARM1- WT or -KO MEF cells. (H) mRNA expression levels of Dnm1l and Carm1 in CARM1-WT or -KO MEF cells. Error bars represent mean ± SD (n = 3). *p < 0.05 and **p < 0.01. (I,J) OCR upon injection of Oligo, CCCP, and R/A into 10T1/2 cells transfected with siDRP1 or mCh-DRP1 (I) or transfected with GFP-CARM1 WT or E266Q mutant (J) for 72 h. Error bars represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are available online for this figure.

Article Snippet: To stain the mitochondria, we used an anti-Alexa FluorTM 488-TOM20 antibody (ProteinTech) or MitoTrackerTM Red CMXRos.

Techniques: RNA Sequencing, Generated, Phospho-proteomics, Injection, Membrane, Software, Western Blot, Expressing, Transfection, Mutagenesis

Journal: Redox biology

Article Title: ROS-mediated cytoplasmic localization of CARM1 induces mitochondrial fission through DRP1 methylation.

doi: 10.1016/j.redox.2024.103212

Figure Lengend Snippet: Fig. 2. CARM1 methylates DRP1 and facilitates its translocation to the mitochondria. (A) OCR of CARM1-WT or -KO MEF cells transfected with mCh-DRP1 for 72 h. Error bars represent mean ± SD (n = 3). ***p < 0.001. (B) Confocal images of MitoTracker™ Red (red), DRP1 (green), and DAPI (blue) in CARM1-WT or -KO MEF cells transfected with an siRNA against DRP1 or plasmid for 72 h. Scale bars, 10 μm. (C) Western blots of mitochondrial and cytoplasmic fractions from 10T1/2 cells transfected with siCARM1 for 24 h and 72 h. (D) In vitro methylation assay using SAM, 10 nM EZM2302, and beads carrying immunoprecipitated CARM1 or DRP1. (E) IP using anti-DRP1 antibody in 10T1/2 cells treated with siCARM1 or 100 nM EZM2302 for 72 h. (F) IP using anti-DRP1 antibody in the mitochondrial and cytoplasmic fractions from 10T1/2 cells. VDAC1 and actin were used as the mitochondrial and cytoplasmic markers, respectively. (G) Western blots of the mito chondrial and cytoplasmic fractions from CARM1-KO MEF cells co-transfected with mCh-DRP1 and GFP-CARM1 (WT or E266Q) for 72 h. (H) PLA with antibodies against DRP1 and ADMA5825, after treatment with digitonin (20 μg/mL, 2 min). Red dots indicate the methylation of DRP1. Mitochondria were stained with an anti- TOM20 antibody conjugated to Alexa Fluor™ 488 (green). Scale bars, 10 μm. (I) Western blots of the mitochondrial and cytoplasmic fractions from 10T1/2 cells treated with 100 nM EZM2302 for 72 h and then released for indicated time. (J) Confocal images of TOM20 (green) in 10T1/2 cells treated with 100 nM EZM2302 for 72 h and then released for indicated time. Mitochondrial interconnectivity was analyzed using ImageJ/FiJi software. Scale bars, 10 μm. Data are presented as mean ± SD (n = 5). **p < 0.01 and ***p < 0.001.

Article Snippet: To stain the mitochondria, we used an anti-Alexa FluorTM 488-TOM20 antibody (ProteinTech) or MitoTrackerTM Red CMXRos.

Techniques: Translocation Assay, Transfection, Plasmid Preparation, Western Blot, In Vitro, Methylation, Immunoprecipitation, Staining, Software

Journal: Redox biology

Article Title: ROS-mediated cytoplasmic localization of CARM1 induces mitochondrial fission through DRP1 methylation.

doi: 10.1016/j.redox.2024.103212

Figure Lengend Snippet: Fig. 5. ROS-mediated cytoplasmic localization of CARM1 reinforces DRP1 methylation and mitochondrial fission. (A-C) Confocal images of TOM20 (green), CARM1 (red), and DAPI (blue) (A), IP of anti-DRP1 antibody (B), and western blots of nuclear, mitochondrial, and cytoplasmic fractions (C) in 10T1/2 cells treated with 100 ng/mL LPS or/and 10 mM NAC for 3 h. Scale bars, 10 μm. (D,E) Double immunostaining for Mff (green) and mCh-DRP1 (red) after treatment with 20 μg/mL digitonin for 2 min (D) and western blots of mitochondrial and cytoplasmic fractions (E) upon introduction of mCh-DRP1 (WT, R403K, R634K, or R639K) into 10T1/ 2 cells. Scale bars, 10 μm. (F–H) Confocal images of TOM20 (green), CARM1 (red), and DAPI (blue) (F), western blots of nuclear, mitochondrial, and cytoplasmic fractions (G), and IP of anti-DRP1 antibody (H) in 10T1/2 cells treated with 10 μM CCCP or/and 10 nM LMB for 3 h. Scale bars, 10 μm. (I) ROS levels measured using FACS in 10T1/2 cells treated with 10 μM CCCP, 100 ng/mL LPS, and/or 10 nM LMB for 3 h. Representative confocal images are shown in Supplementary Fig. 5D.

Article Snippet: To stain the mitochondria, we used an anti-Alexa FluorTM 488-TOM20 antibody (ProteinTech) or MitoTrackerTM Red CMXRos.

Techniques: Methylation, Western Blot, Double Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome

doi: 10.1007/s00018-018-2771-6

Figure Lengend Snippet: The TRAP-activated PLT extracellular vesiculome contained “free” mitochondria and vesicles containing mitochondria. LSCM images of membrane and mitochondria of a resting PLTs and b the PLT extracellular vesiculome labeled with Green-CellMask™ and Red-TOM20ab. PLTs labeled with Green-CellMask™ and Red-MitoTracker c before and d after activation; e FC analysis confirmed that PLT extracellular vesiculome contain mitochondria and showed that a portion of PEVs positive for mitochondrial markers (TOM20, mitotracker) also carry the autophagosomal marker LC3. The bar graph shows % of Mitotracker+PEVs, TOM20+PEVs, LC3+PEVs, TOM+20LC3+PEVs, CD41a+PEVs, CD41a+TOM20+PEVs, CD41a+LC3+PEVs in total PLT extracellular vesiculome labeled by DHPE (DHPE+PEVs), see SI Fig. 6 for representative scatter plots; f Cryo-TEM image of a mitochondrion (double bilayer) [19] in L-PEVs; g, h FESEM images showing large membrane blisters that resemble apoptotic bodies; i cryo-TEM image of a large vesicle containing organelles present in the extracellular vesiculome; j graphic illustration of a proposed mechanism for organelle sequestration into vesicles: PLT activation induces cytoskeleton contraction [49] with consequent PM detachment with increase in hydrostatic pressure toward PM [50, 109] causing peripheralization of organelles [81]. PLTs were activated with TRAP (20 µmol/L TRAP for 30 min, RT with gentle agitation) in Tyrodes buffer (a–d, h) or plasma (f, g); results are representatives of three independent experiments. Error bars indicate SD

Article Snippet: Anti-human Tom20 (F-10) Alexa Fluor 488-conjugated (#sc-17764 AF488) or Alexa Fluor 647-conjugated (# sc-17764 AF647) were from Santa Cruz; goat anti-rabbit IgG H&L secondary antibody (Alexa Fluor ® 555; #ab150078) was from Abcam; Alexa Fluor ® 488 anti-human CD62P (P-Selectin) antibody (#304916) was from Biolegend; FITC mouse anti-human CD41a (clone HIP8; #555466), PE mouse anti-human CD62P (clone AK-4; #555524), PE annexinV (#556422), PE conjugated mouse IgG1 (clone MOPC-21; isotype control; #559320) and FITC-conjugated mouse IgG1 (clone MOPC-21; isotype control; # 555748) were from BD Pharmingen; uranyl acetate (#19481) and lead citrate (#19314) were from TedPella; osmium tetroxide (#19150), EMBed-812 embedding media (#14120), cacodylate buffer (#11652), paraformaldehyde (PFA; #15710) and glutaraldehyde (GTA; #16220) were from Electron Microscopy Science; 1× Tris-BSA buffer, pH 7.3 (#AAR020A) Aniara Diagnostica LLC, normal plasma (#FRNCP0125) Affinity Biologicals Inc, TF from Dade Innovin, Z-Gly-Gly-Arg-AMC.

Techniques: Membrane, Labeling, Activation Assay, Marker, Gentle, Clinical Proteomics

Journal: Cell Genomics

Article Title: In vivo CRISPR screening directly targeting testicular cells

doi: 10.1016/j.xgen.2024.100510

Figure Lengend Snippet: RD3 modulates mitochondrial spatial distribution under ciliogenesis-induction-derived oxidative stress (A) RD3 and mitochondria co-localization study. SPOT-tagged RD3 SH-SY5Y cells stained with anti-RD3 (red) and Alexa 488-conjugated Tomm20 (green) antibodies with DAPI (white) and imaged by confocal microscope. Scale bar: 5 μm. Cell: SH-SY5Y. (B and E) Working hypothesis. (C) Quantification of ciliogenesis frequency. Primary cilium frequency quantified using filtered sections (30–50 cells per section). RD3 -wild type (WT): 803 cells (navy); KO: 1,269 cells (cobalt blue); and overexpression (OE): 1,141 cells (light green) are shown as a violin plot. Each frequency per section is shown as white dots. p value: an unpaired t test. p < 0.05: orange. Experiments were repeated three times (n = 3). (D) Cilium length quantification. Primary cilium length quantified by Nikon NIS-Element software using filtered sections. RD3 -WT: 71 cilia (navy); KO: 106 cilia (cobalt blue); and OE: 72 cilia (light green) are shown as a cumulative plot. Statistical analysis was conducted as described in (C). (F) Mitochondria distribution imaging. Ciliogenesis was induced by serum starvation. Cells were stained with anti-α-tubulin (magenta), Alexa 488-conjugated Tomm20 (green), and Alexa 647-conjugated γ-tubulin (yellow) antibodies and DAPI (white), imaged by confocal microscope. Scale bar: 20 μm. (G) Mitochondrial distribution quantification. The mitochondria distribution index calculated at 5 different spots is defined by the distance from the γ-tubulin signal. Quantification was performed by around 30 cells per replicate. Quantifications were conducted three times (n = 3). p value: an unpaired t test. p < 0.05: orange. (H) Mitochondrial distribution differences. Distribution difference between RD3 -WT and KO is shown as barplot. p value: an unpaired t test. (I) Mitochondrial H 2 O 2 evaluation. MitoPY1 and Hoechst33342 signals measured by plate reader. Raw MitoPY1 signal was normalized by Hoechst33342, multiplied by 1 × 10 4 , and shown as a bar plot. p value: an unpaired t test. p < 0.05: orange. Experiments were repeated five times (n = 5). (J) Summary of Rd3-mitochondria spatial dynamics under cilium-induction-oriented oxidative stress.

Article Snippet: Alexa Fluor 488 Anti-Tomm20 antibody - Mitochondrial Marker , Abcam , Catalog #: ab205486 Clone: EPR15581-39 RRID: AB_2943509.

Techniques: Derivative Assay, Staining, Microscopy, Over Expression, Software, Imaging

Journal: Cell Genomics

Article Title: In vivo CRISPR screening directly targeting testicular cells

doi: 10.1016/j.xgen.2024.100510

Figure Lengend Snippet:

Article Snippet: Alexa Fluor 488 Anti-Tomm20 antibody - Mitochondrial Marker , Abcam , Catalog #: ab205486 Clone: EPR15581-39 RRID: AB_2943509.

Techniques: Marker, CRISPR, Knock-Out, Recombinant, Cell Counting, Staining, Whole Genome Amplification, Software