Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Multidirectional fatty acid (FA) trafficking pathways between lipid droplets (LDs) and mitochondria. Terms: FAO, fatty acid oxidation; NG497, an ATGL inhibitor; Triacsin C, an inhibitor of long FA acyl-CoA synthetase. (B) BODIPY 493/503-labeled LDs detected via confocal microscopy before and after 6-h incubation with 10 µM Triacsin C in wildtype (WT) and OPA1 knockout (KO) U2OS cells treated with 100 µM oleic acid (OA) overnight. Maximal intensity projected (MIP) confocal images from whole cells are shown. Dashed lines mark cell boundary. (C) Quantification of BODIPY 493/503-positive LD content as described in (B). Mean ± standard deviation from three independent experiments are shown (total of 99–161 cells). n.s., no significance, **P ≤ 0.001, as assessed by one-way ANOVA. (D) Quantification of monodansylpentane (MDH)-positive LD content in WT, OPA1 KO, and OPA1-expressing OPA1 KO U2OS cells treated with 100 µM OA overnight followed by 10 µM Triacsin C incubation for 6 h. Mean ± standard deviation from three independent experiments are shown (total of 81–115 cells). n.s., no significance, **P ≤ 0.01, *P ≤ 0.05, as assessed by one-way ANOVA. (E) Subcellular localization of TopFluor (TF)-OA and TOM20-Halo (JF646) detected via confocal microscopy in WT and OPA1 KO U2OS cells treated with 20 µM DGAT1 and DGAT2 inhibitors. MIP confocal images from three axial slices (∼0.6 µm total thickness) are shown. (F) Quantification of the intensity of peripheral mitochondria (mito) of TF-OA as described in (E) and in cells pretreated with 10 µM Triacsin C, 1 µM BODIPY-linoleic acid (LA), or 1 µM NBD-arachidonic acid (AA). Mean ± standard deviation from three independent experiments are shown (total of 38–50 cells). n.s., no significance, ****P ≤ 0.0001, *P ≤ 0.05, as assessed by one-way ANOVA. (G) Scintillation counts per minute (CPM) for complete 14 C-OA oxidation in WT and OPA1 KO U2OS cells under control and Triacsin C-treated conditions. Mean ± standard deviation from three independent experiments are shown. n.s., no significance, ***P ≤ 0.001, *P ≤ 0.05, as assessed by one-way ANOVA. (H) Concentration of cholesterol-ester (CE), diacylglycerol (DAG), triglyceride (TAG), and monoacylglycerol (MAG) in steady-state WT and OPA1 KO U2OS cells determined via liquid chromatography–mass spectrometry. Mean ± standard deviation from four replicates are shown. n.s., no significance, ****P ≤ 0.0001, as assessed by unpaired t -test. (I) Variance in acyl-carnitine levels across acyl-chain length in steady-state WT and OPA1 KO U2OS cells determined using liquid chromatography–mass spectrometry. Mean ± standard deviation from four replicates are shown. n.s., no significance, ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, as assessed by unpaired t -test.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Confocal Microscopy, Incubation, Knock-Out, Standard Deviation, Expressing, Control, Concentration Assay, Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Diagram illustrating the generation of OPA1 knockout (KO) U2OS cells via CRISPR-genome editing. Sequencing data validating OPA1 KO is shown in the top right, and deleted OPA1 genome sequences are represented as dashed lines highlighted in red. (B) Western blot analysis of OPA1, ATGL, DGAT1, and GAPDH in wildtype (WT) and OPA1 KO U2OS cells -/+ overnight 100 µM oleic acid (OA) treatment. (C) Immunostaining of endogenous OPA1 and TOM20 in WT and OPA1 KO U2OS cells detected by confocal microscopy. Maximal intensity projected confocal images from whole cells with min-max intensity range (gray boxes) are shown. (D) Western blot analysis of OPA1 and GAPDH in HeLa cells transfected with scramble siRNA (siCtrl) or OPA1 siRNA. (E) Western blot analysis of OPA1 and GAPDH in WT and OPA1 KO mouse embryonic fibroblasts (MEFs) -/+ overnight 100 µM OA treatment. (F) Western blot analysis of HSL, CGI-58, and GAPDH in WT and OPA1 KO U2OS cells -/+ overnight 100 µM OA treatment.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Knock-Out, CRISPR, Sequencing, Western Blot, Immunostaining, Confocal Microscopy, Transfection

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) BODIPY 493/503-positive area in U2OS cells treated with 100 µM oleic acid (OA) overnight followed by 10 µM Triacsin C incubation in the presence of 10 µM NG497 (NG) or 5 µM Lalistat 2 (LALi) for 6 h. Mean ± standard deviation from three independent experiments are shown (total of 129–148 cells). For statistics in panels A–E, n.s., no significance, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, as assessed by one-way ANOVA. (B) BODIPY 493/503-positive area in U2OS cells treated with 100 µM OA overnight followed by 6-h co-incubation of 10 µM Triacsin C and 10 µM H89. (C) BODIPY 493/503-positive area in HeLa cells transfected with scramble siRNA (siCtrl) or OPA1 siRNA (siOPA1) treated with 100 µM OA overnight before 6-h incubation with 10 µM Triacsin C. Mean ± standard deviation from three independent experiments are shown (total of 107–122 cells). (D) BODIPY 493/503-positive area in WT and OPA1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with 100 µM OA overnight before 6-h incubation with 10 µM Triacsin C. Mean ± standard deviation from three independent experiments are shown (total of 83–105 cells). (E) BODIPY 493/503-positive area in WT and OPA1 KO U2OS cells treated with 100 µM OA overnight followed by 20-h incubation in glucose-free Dulbecco’s Modified Eagle Medium. Mean ± standard deviation from three independent experiments are shown (total of 79–87 cells). (F and G) Levels of (F) glycerophospholipids and (G) sphingolipids in steady-state WT and OPA1 KO U2OS cells determined using liquid chromatography–mass spectrometry. Mean ± standard deviation from four replicates are shown. For statistics in panels F and G, n.s., no significance, ***P ≤ 0.001, **P ≤ 0.01, as assessed by unpaired t -test. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PI, phosphatidylinositol; CER, ceramide; DCER, dihydroceramide; HCER, hexosylceramide; LCER, lactosylceramide; SM, sphingomyelin.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Incubation, Standard Deviation, Transfection, Knock-Out, Modification, Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Localization of OPA1-YFP (lentiviral), mitochondria (labeled with MitoTracker Deep Red), and lipid droplets (LDs; labeled with MDH) in U2OS cells -/+ overnight 100 µM oleic acid (OA) treatment. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. Cyan arrowheads indicate area with LDs. (B) Relative enrichment of MitoTracker and OPA1 on LDs from (A) and in cells treated with 100 µM linoleic acid (LA) or arachidonic acid (AA) overnight. Mean ± standard deviation from 3–5 independent experiments are shown (total of 43–74 cells). n.s., no significance, ****P ≤ 0.0001, assessed by one-way ANOVA. (C) Subcellular localization of endogenous OPA1 on mitochondria (anti-TOM20) and LDs labeled by BODIPY 493/503 in an OA-treated U2OS cell monitored with confocal microscopy. Sum of confocal images from five axial slices (∼1.2 µm in total thickness) are shown. (D) Relative intensity profiles of OPA1, TOM20, and BODIPY measured along the white-dashed arrow from lower left panel in (C). (E) Western blot analysis of endogenous OPA1, PLIN 2 (an LD protein), and PHB 2 (a mitochondrial inner membrane protein) in sucrose-gradient cellular fractionations from U2OS cells treated with 500 μM OA. (F) Correlative confocal-scanning electron microscopy (SEM) images of OPA1 on LDs and in mitochondria in OPA1-mhYFP–expressing U2OS cells treated with 100 µM OA overnight. Confocal and SEM images from a single axial slice are shown. EM, electron microscopy. Inset outlined in blue is shown in . (G) Subcellular distribution of inducible OPA1 (iOPA1)-YFP on LDs (BODIPY 665/676) and in mitochondria (MitoTracker Red) in OPA1 KO U2OS cells following treatment with 4 µg/mL doxycycline for 4 h. Confocal images from a single axial slice are shown. Cyan arrowheads indicate regions containing LDs. (H) Fraction of cells with iOPA1 localized to indicated organelle as described in (G). Mean ± standard deviation from three independent experiments are shown. (I) Localization of OPA1-YFP, mitochondria (MitoTracker Deep Red), LDs (MDH) in U2OS cells treated with 100 µM OA and 20 µM FCCP (an uncoupler of mitochondria oxidative phosphorylation) overnight detected by confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. (J) Relative enrichment of OPA1 on LDs in U2OS cells treated with 100 µM OA -/+ 20 µM FCCP for overnight in (I). Mean ± standard deviation from four independent experiments are shown (total of 57–73 cells). **P ≤ 0.01, as assessed by unpaired t -test.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Standard Deviation, Confocal Microscopy, Western Blot, Membrane, Electron Microscopy, Expressing, Phospho-proteomics

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Localization of OPA1-YFP (lentiviral), mitochondria (labeled with MitoTracker Deep Red), and lipid droplets (LDs, labeled with MDH) in U2OS cells treated with 100 µM linoleic acid (LA) or 100 µM arachidonic acid (AA) overnight. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (B) Localization of OPA1-YFP, mitochondria (MitoTracker Deep Red), and LD (MDH) in HeLa and Huh7 cells treated with 100 µM oleic acid (OA) overnight, detected with confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. Cyan arrowheads indicate area with LDs. ( C ) Inset from the correlative confocal-scanning electron microscopy (SEM) image in , outlined in blue, showing OPA1-mhYFP localization in mitochondria in a U2OS cell treated with 100 µM OA overnight. (D) Subcellular localization of endogenous OPA1 (anti-Opa1) on mitochondria (anti-TOM20) and LDs (BODIPY 493/503) in a U2OS cell treated with 100 µM OA and 20 µM FCCP (an uncoupler of mitochondria oxidative phosphorylation) overnight and monitored via confocal microscopy. Sum of confocal images from five axial slices (∼1.2 µm total thickness) are shown. (E) Relative intensity profiles of OPA1, TOM20, and BODIPY measured from lower right panel in (D), indicated by white-dashed arrows. (F) Properties of representative mitochondrial targeting sequences (MTSs) for mitochondrial protein import. Asterisk indicates the predicted import velocity of the OPA1 MTS. Max μH, maximal helical hydrophobic moment. (G) Correlation between the amphiphilicity of MTS and the protein import velocity from (F). (H) Localization of truncated YFP-tagged OPA1 fragments (frag.), mitochondria (labeled with TOM20-Halo; JF646), and LDs (MDH) in U2OS cells treated with 100 µM OA overnight and detected by confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Confocal Microscopy, Electron Microscopy, Phospho-proteomics

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Constructs expressing full-length and truncated OPA1, as well as summary of their primary subcellular localizations. Amino acid number and protein domains are indicated. MTS, mitochondria targeting sequence; GED, GTPase effector domain; LD, lipid droplet; mito, mitochondria. (B) Localization of OPA1 ΔMTS -YFP, mitochondria (labeled with MitoTracker Deep Red), and lipid droplets (LDs; labeled with MDH) in U2OS cells treated with 100 µM oleic acid (OA) overnight and detected by confocal microscopy. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (C) Relative enrichment of OPA1 ΔMTS from (B) and OPA1 on LDs in U2OS cells treated with 100 µM OA overnight. Mean ± standard deviation from three independent experiments are shown (total of 48–55 cells). ****P ≤ 0.0001, as assessed by unpaired t -test. (D) Amphipathic helix structure of exon 4 as predicted by AlphaFold. Cyan and yellow indicate polar and non-polar amino acids, respectively, and black arrows indicate location of residues for mutagenesis. (E) Localization of YFP-tagged exon 4 in MDH-stained U2OS cells treated with 100 µM OA overnight and detected by confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. (F) Correlative confocal-scanning electron microscopy (SEM) images of exon 4 on LDs in exon 4-mhYFP expressing U2OS cells treated with 100 µM OA overnight. Confocal and SEM images from a single axial slice are shown. (G) Localization of exon 4 mutants relative to LDs (MDH) in U2OS cells treated with 100 µM OA overnight and detected by confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. LFAA, a double mutation of L156 and F169 to alanines. (H) Relative enrichment of exon 4 and mutants (described in E and G), as well as PLIN 2-GFP on LDs in U2OS cells treated with 100 µM OA overnight. Mean ± standard deviation from two or three independent experiments are shown (total of 30–52 cells). ****P ≤ 0.0001, ***P ≤ 0.001, as assessed by one-way ANOVA. (I) Localization of OPA1 and OPA1 LFAA , mitochondria (MitoTracker Deep Red), and LD (MDH) in U2OS cells treated with 100 µM OA overnight and detected by confocal microscopy. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. (J) Relative enrichment of OPA1 and OPA1 LFAA from (I) on LDs in cells treated with 100 µM OA overnight. Mean ± standard deviation from three independent experiments are shown (total of 63–65 cells). ****P ≤ 0.0001, as assessed by one-way ANOVA.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Construct, Expressing, Sequencing, Labeling, Confocal Microscopy, Standard Deviation, Mutagenesis, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Percentage of OPA1 exon 4 inclusion and exclusion, representing OPA1 isoform 1 and isoform 2, respectively, across various human tissues analyzed from the Genotype-Tissue Expression (GTEx) database. Raw data and median values with quartile ranges are shown. (B) Profiling of OPA1 exon 4 inclusion (incl.) and exclusion (excl.) from cDNA of HeLa and U2OS cells. (C) Localization of OPA1-YFP or OPA1 iso2 -YFP, mitochondria (labeled with MitoTracker Deep Red), and lipid droplets (LDs; labeled with MDH) in HeLa cells treated with 100 µM oleic acid (OA) -/+ 20 µM FCCP (an uncoupler of mitochondria oxidative phosphorylation) overnight and detected by confocal microscopy. Maximal intensity projected confocal images from four axial slices (∼1 µm in total thickness) are shown. (D) Relative enrichment of OPA1 and OPA1 iso2 on LDs as described in (C). Mean ± standard deviation from three-four independent experiments are shown (total of 31–42 cells). Yellow dashed line indicates the relative enrichment of OPA1 on LDs in U2OS cells as described in . n.s., no significance, ***P ≤ 0.001, *P ≤ 0.05, as assessed by one-way ANOVA. (E) BODIPY-positive LD content in siCtrl and siOPA1 4-5 junc transfected HeLa cells treated with 100 µM OA overnight followed by incubation with 10 µM Triacsin C for 6 h. Mean ± standard deviation from four independent experiments are shown (total of 107–122 cells). n.s., no significance, **P ≤ 0.01, as assessed by one-way ANOVA.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Expressing, Labeling, Phospho-proteomics, Confocal Microscopy, Standard Deviation, Transfection, Incubation

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Localization of OPA1 iso2 -YFP, mitochondria (labeled with MitoTracker Deep Red), lipid droplets (LDs; labeled with MDH) in U2OS cells treated with 100 µM oleic acid (OA) -/+ 20 µM FCCP (an uncoupler of mitochondria oxidative phosphorylation) overnight detected by confocal microscopy. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (B) Relative enrichment of OPA1 iso2 as described in (A) and OPA1 on LDs in U2OS cells treated with 100 µM OA overnight. Mean ± standard deviation from three independent experiments are shown (total of 43–51 cells). n.s., no significance, ****P ≤ 0.0001, as assessed by one-way ANOVA. (C) Subcellular distribution of inducible OPA1 iso2 (iOPA1 iso2 )-YFP on LDs (BODIPY 665/676) and mitochondria (MitoTracker Red) in OPA1 knockout (KO) U2OS cells following 4 µg/mL doxycycline treatment for 4 h. Confocal images from a single axial slice are shown. (D) Fraction of cells with iOPA1 iso2 localized to indicated organelle as described in (C). Mean ± standard deviation from three independent experiments are shown. (E) Design of an siRNA targeting the OPA1 exon 4–exon 5 junction for selective depletion of isoform 1. (F and G) Levels of (F) OPA1 isoform-specific mRNA or (G) protein in U2OS cells transfected with the indicated siRNAs. Mean ± standard deviation from two or three independent experiments are shown in (F). (H) Relative BODIPY-positive area indicating LD content in siCtrl and siOPA1 4-5 junc transfected U2OS cells treated with 100 µM OA overnight followed by 10 µM Triacsin C incubation for 6 h. Mean ± standard deviation from three independent experiments are shown (total of 90–101 cells). n.s., no significance, ***P ≤ 0.001, as assessed by one-way ANOVA.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Phospho-proteomics, Confocal Microscopy, Standard Deviation, Knock-Out, Transfection, Incubation

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Western blot analysis of endogenous ATGL, HSL, and PLIN 2 in sucrose-gradient cellular fractionations from wildtype (WT) and OPA1 knockout (KO) U2OS cells treated with 500 μM oleic acid (OA). (B) ATGL and HSL abundance in lipid droplet (LD) fractions normalized to PLIN2 as described in (A). Mean ± standard deviation from three independent experiments are shown. **P ≤ 0.01, *P ≤ 0.05, as assessed by unpaired t -test. (C) Localization of ATGL S47A-Halo and LDs (labeled with BODIPY-493/503) in WT and OPA1 KO U2OS cells treated with 100 µM OA overnight. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (D) Relative enrichment of endogenous PLIN 2 (detected by immunostaining), ATGL S47A -Halo, and mApple-CGI-58 on LDs from , and , respectively. Mean ± standard deviation from three independent experiments are shown (total of 72–74 cells for ATGL S47A -Halo; total of 50-52 cells for PLIN 2, and total of 34–46 cells for mApple-CGI-58). n.s., no significance, **P ≤ 0.01, as assessed by unpaired t -test. (E) Relative enrichment of ATGL S47A -Halo on LDs in OPA1 KO U2OS cells and OPA1 KO cells reconstituted with lenti-OPA1-YFP or lenti-OPA1 LFAA -YFP. Mean ± standard deviation from three independent experiments are shown (total of 56–60 cells). For statistics in panels E and F, n.s., no significance, **P ≤ 0.01, *P ≤ 0.05, as assessed by one-way ANOVA. (F) MDH-positive LD content in WT and OPA1 KO U2OS cells, as well as OPA1 KO cells reconstituted with lenti-OPA1-YFP or lenti-OPA1 LFAA -YFP. Cells were treated with 100 µM OA overnight followed by 6-h incubation with 10 µM Triacsin C. Mean ± standard deviation from three independent experiments are shown (total of 71–97 cells).

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Western Blot, Knock-Out, Standard Deviation, Labeling, Immunostaining, Incubation

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Localization of endogenous PLIN 2 on lipid droplets (LDs) labeled with BODIPY-493/503 in wildtype (WT) and OPA1 knockout (KO) U2OS cells treated with 100 µM oleic acid (OA) overnight. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (B) Localization of mApple-CGI-58 and LDs labeled with BODIPY-493/503 in WT and OPA1 KO U2OS cells treated with 100 µM OA overnight. MIP confocal images from four axial slices (∼1 µm total thickness) are shown. (C–E) Two-dimensional ‘mitochondria analyzer’ analysis of mitochondrial morphology and connectivity measuring (C) form factor, (D) total branch length, and (E) branch junction in MitoTracker DeepRed–stained WT and OPA1 KO U2OS cells -/+ overnight 100 µM OA treatment. Mean ± standard deviation from three independent experiments are shown (total of 74–100 cells). For panels C–E, n.s., no significance, ****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05, as assessed by one-way ANOVA.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Knock-Out, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) A schematic illustrating two-dimensional (2D) analysis of mitochondrial morphology and connectivity using the Fiji plugin, ‘Mitochondria-Analyzer’. Representative MitoTracker DeepRed images (maximal intensity projected confocal images from three axial slices; 0.6 µm in thickness) from OPA1 knockout (KO) U2OS cells and the corresponding analyses are shown. (B–D) Two-dimensional analysis of mitochondrial morphology and connectivity measuring (B) form factor, (C) total branch length, and (D) branch junction in MitoTracker DeepRed-stained OPA1 KO U2OS cells and OPA1 KO U2OS cells reconstituted with lenti-OPA1-YFP or lenti-OPA1 LFAA -YFP. Mean ± standard deviation from three or four independent experiments are shown (total of 80–133 cells). n.s., no significance, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, as assessed by one-way ANOVA. Black asterisks represent comparisons with OPA1 KO, and purple asterisks represent the indicated comparison.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Knock-Out, Staining, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

doi: 10.64898/2026.05.07.723579

Figure Lengend Snippet: (A) Amphipathic helix structures and hydrophobicity distributions of exon 4 and exon 4 S158N, predicted by AlphaFold and illustrated in ChimeraX. Cyan and yellow indicate polar and non-polar amino acids, respectively, and arrows denote locations of indicated residues. (B) Localization of YFP tagged OPA1 or OPA1 S158N (lentiviral), mitochondria (labeled with MitoTracker Deep Red), and lipid droplets (LDs; labeled with MDH) in U2OS cells treat with 100 µM oleic acid (OA) overnight. Maximal intensity projected (MIP) confocal images from four axial slices (∼1 µm total thickness) are shown. (C) Relative enrichment of OPA1 on LDs in U2OS cells as described in (B). Mean ± standard deviation from three independent experiments are shown (total of 50-60 cells). **P ≤ 0.01, assessed by unpaired t -test. (D) MDH-positive LD content in wildtype (WT) and OPA1 KO U2OS cells, and in OPA1 KO cells reconstituted with lenti-OPA1-YFP or lenti-OPA1 S158N -YFP. Cells were treated with 100 µM OA overnight followed by incubation with 10 µM Triacsin C 6 h. Mean ± standard deviation from three independent experiments are shown (total of 63-83 cells). (E–G) Two-dimensional analysis of mitochondrial morphology and connectivity via measuring (E) form factor, (F) total branch length, and (G) branch junction in MitoTracker DeepRed-stained OPA1 KO U2OS cells and OPA1 KO cells reconstituted with lenti-OPA1-YFP or lenti-OPA1 S158N -YFP. Mean ± standard deviation from 3-4 independent experiments are shown (total of 55-83 cells). For panels D-G, n.s., no significance, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, assessed by one-way ANOVA. (H–J) Clinical data was mined from the St. Jude LIFE database to assess (H) body fat percentage, (I) blood triacylglycerol levels, and (J) blood cholesterol levels in patients with different S158N genotypes. (K) Proposed model of OPA1’s functions and impacts on LDs and mitochondria depending on its alternative targeting and its implication in human metabolic outcomes. OPA1-mediated lipase recruitment to LDs is indicated with a dashed arrow. Abbreviations: FAO, fatty acid oxidation; MTS, mitochondria targeting sequence.

Article Snippet: U2OS cells (HTB-96), HeLa (CCL-2) cells, and OPA1 KO MEFs (CRL-2995) were purchased from American Type Culture Collection (ATCC); Huh7 (JCRB0403) cells were purchased from the Japanese Cancer Research Bank.

Techniques: Labeling, Standard Deviation, Incubation, Staining, Sequencing

Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Viability values (%) of 3T3 cells exposed to different concentrations of E. faecalis phage Max and E. faecium phage Zip for 24 h ( A ) Phage lysate; ( B ) Phages purified by PEG/NaCl precipitation. Viability was calculated as percentage of negative control (3T3 cells without phages). Error bars represent standard deviations from three independent experiments performed in duplicate.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Purification, Negative Control

Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Efficacy of phages against bacteria colonizing 3T3 cells. ( A ) Concentration of viable bacterial cells; and ( B ) number of 3T3 cells in control (without treatment) and after 6 and 24 h of phage treatment. Error bars represent standard deviations from three independent experiments performed in duplicate. *Statistically significant (p < 0.05) conditions between control and test assays.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Bacteria, Concentration Assay, Control

Journal: Journal of cell science

Article Title: Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway.

doi: 10.1242/jcs.062919

Figure Lengend Snippet: Fig. 3. Cav1 negatively regulates Rac1- mediated cell spreading, but not Rac1 targeting. (A)Immunostaining for endogenous Rac1 in wild-type (wt) and Cav1–/– MEFs shows localization of Rac1 at FAs (arrows), marked by pY118 paxillin, even in the absence of Cav1. (B)Rac1 C-terminal interaction with b-Pix was analyzed in the absence or presence of Cav1 using lysates from wild-type (wt) and Cav1–/– MEFs. The association of Rac1 with b- Pix is independent of Cav1. (C)Staining for F- actin underscores the cell spreading and loss of polarity of HeLa cells transfected with Cav1 siRNA on fibronectin-coated glass coverslips. Scale bar: 20m. The lower polarized cell in which Cav1 expression is not reduced serves as a positive control. (D)Cell spreading of wild- type (wt) and Cav1–/– MEFs in the presence or absence of the Rac1 inhibitor EHT1864 (50M) was analyzed by ECIS on fibronectin- coated gold electrodes and depicted as normalized resistance. (E)Phase-contrast images of Cav1–/– MEFs spread on fibronectin for 30 minutes or 3 hours with or without EHT1864 (50M). Scale bar: 20m. These data show that the increased spreading in the Cav1–/– MEFs is Rac1 dependent.

Article Snippet: Cav1–/– knockout (ATCC-CRL-2752) and parental wild-type (ATCC-CRL-2753) MEFs were obtained from ATCC and cultured in DMEM containing 10% heat-inactivated FCS (Bodinco), 2 mM Lglutamine and penicillin-streptomycin.

Techniques: Immunostaining, Staining, Transfection, Expressing, Positive Control

Journal: Journal of cell science

Article Title: Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway.

doi: 10.1242/jcs.062919

Figure Lengend Snippet: Fig. 4. Loss of Cav1 increases total and activated Rac1 levels. (A)Rac1 activation was assayed by biotinylated Pak-Crib peptide-based pull down using lysates of control cells and HeLa cells transfected with Cav1 siRNA. The bar diagram depicts the relative increase in Rac1 expression and activation levels in cells treated with Cav1 siRNA compared with control cells, as determined by quantification of western blots (n3). Loss of Cav1 is associated with an approximately twofold increase in total and activated levels of Rac1. (B)Rac1 activation and expression were assayed by biotinylated Pak-Crib peptide-based pull down in lysates of wild- type (wt) and Cav1–/– MEFs. The bar diagram depicts the relative increase in Rac1 expression and activation levels in Cav1-depleted Cav1–/– fibroblasts compared with parental cells, as determined by quantification of blots (n3). Like siRNA-based depletion of Cav1, loss of Cav1 in Cav1–/– MEFs is associated with an approximately twofold increase in total and activated levels of Rac1. (C)SDS lysates of control cells and cells transfected with Cav1 siRNA were analyzed for protein levels of Rac1, CDC42 and RhoA. Rac1 levels in SDS lysates were also determined in HUVEC cells transfected with Cav1 siRNA and in Cav1–/– MEFs. (D) Endogenous ubiquitylated Rac1 was isolated by cobalt-bead-based pull down from lysates of control cells and HeLa cells transfected with Cav1 siRNA co- transfected with 6His-myc-tagged ubiquitin. Two exposures of the western blot are shown to underscore the increase in mono-ubiquitylated endogenous Rac1 (short exposure, left) and the reduction in polyubiquitylated Rac1 (long exposure, right) in cells transfected with the Cav1 siRNA. The relative differences in mono- and poly-ubiquitylation were quantified by densitometry (n3) and are indicated in the bar graphs. In addition, the sizes of the various ubiquitylated forms of Rac1 (Rac1 ~22 kDa; Rac1-Ub ~30 kDa; Rac1-Ub2 ~38 kDa; Rac1-Ub3 ~46 kDa and Rac1-Ub4 ~54 kDa) are indicated. (E)Analysis of adhesion-dependent mono- ubiquitylation of endogenous Rac1 in HeLa cells. Prior to lysis and isolation of ubiquitylated proteins, cells transfected with 6His-myc-tagged ubiquitin were either seeded on fibronectin for 2 hours or kept in suspension. Western blotting following isolation of ubiquitylated proteins was used to detect mono-ubiquitylated Rac1 (short exposure, left) and polyubiquitylated Rac1 (long exposure, right), as detected by immunostaining with a Rac1 antibody.

Article Snippet: Cav1–/– knockout (ATCC-CRL-2752) and parental wild-type (ATCC-CRL-2753) MEFs were obtained from ATCC and cultured in DMEM containing 10% heat-inactivated FCS (Bodinco), 2 mM Lglutamine and penicillin-streptomycin.

Techniques: Activation Assay, Control, Transfection, Expressing, Western Blot, Isolation, Ubiquitin Proteomics, Lysis, Suspension, Immunostaining

Journal: Life Science Alliance

Article Title: Amino acid–dependent TSC2 dephosphorylation by lysosome–PP2A regulates mTORC1 signaling transduction

doi: 10.26508/lsa.202503206

Figure Lengend Snippet: (A) p18Rev and TSC2-KO MEFs (#1 and #2) were exposed to AA deprivation and subsequent AA and Ins treatment. Cell lysates were analyzed for TSC2 and AKT phosphorylation using anti-phospho-TSC2 (P-T1462 and P-S939) and anti-phospho-AKT (P-T308 and P-S473) antibodies, respectively. The expression levels of TSC2 and AKT in the cell lysates are also shown. (B) MEFs and HEK293 cells were transfected with either a control siRNA or siRNAs specific for TSC2, then subjected to AA starvation, followed by treatment with AA and Ins for 20 min. Cell lysates were analyzed by immunoblotting. (C) Immunofluorescence analysis of p18Rev and TSC2-KO (#1 and #2) MEFs and si-Control– or si-TSC2–treated MEFs cultured in growth medium supplemented with AAs. TSC2, green; LAMP1, red. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. Fluorescence signals of TSC2 were abolished in TSC2-KO cells (#1 and #2). (D) Immunofluorescence analysis of si-Control– or si-TSC2–transfected HEK293 cells cultured in growth medium supplemented with AAs. TSC2, green; DAPI, blue. Scale bars, 10 μm. (E, F) Phosphorylation levels of AKT at T308 and S473, TSC2 at T1462, and S6K at T389 monitored in p18Rev cells treated with a combination of Ins and AA for the indicated times (10 min to 24 h) (E) and 3–60 min (F). p18Rev cells were treated with AA and Ins as shown in . Cell lysates were analyzed by Western blotting using the indicated antibodies. Source data are available for this figure.

Article Snippet: Normal/parental MEF , p18-KO, p18Rev, p18/Rheb-dKO, and its derivative MEFs , HEK293, and HeLa cells were cultured in DMEM (4.5 g/liter glucose [08458-16; Nacalai Tesque] for MEFs; 1.0 g/liter glucose [08456-36; Nacalai Tesque] for HEK293 and HeLa) supplemented with 10% (vol/vol) fetal bovine serum (NICHIREI BIOSCIENCES INC) and penicillin/streptomycin (09367-34; Nacalai Tesque) at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Phospho-proteomics, Expressing, Transfection, Control, Western Blot, Immunofluorescence, Cell Culture, Staining, Fluorescence

Journal: Life Science Alliance

Article Title: Amino acid–dependent TSC2 dephosphorylation by lysosome–PP2A regulates mTORC1 signaling transduction

doi: 10.26508/lsa.202503206

Figure Lengend Snippet: Phosphorylation levels of AKT at T308 and TSC2 at T1462 monitored in p18Rev and p18-KO MEFs subjected to AA deprivation and subsequent Ins treatment, alone or in combination with AA. Cells were cultured in minimal medium without AA for 1 h and then incubated with medium containing Ins (10 μg/ml) alone or in combination with AA for the indicated time. Cell lysates were analyzed by Western blotting using the indicated antibodies. Note that trial-1 bands of P-TSC2 (P-T1462) and P-AKT (P-T308) are the same ones in . Source data are available for this figure.

Article Snippet: Normal/parental MEF , p18-KO, p18Rev, p18/Rheb-dKO, and its derivative MEFs , HEK293, and HeLa cells were cultured in DMEM (4.5 g/liter glucose [08458-16; Nacalai Tesque] for MEFs; 1.0 g/liter glucose [08456-36; Nacalai Tesque] for HEK293 and HeLa) supplemented with 10% (vol/vol) fetal bovine serum (NICHIREI BIOSCIENCES INC) and penicillin/streptomycin (09367-34; Nacalai Tesque) at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Phospho-proteomics, Cell Culture, Incubation, Western Blot