Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) Live confocal microscopy imaging of U2OS stably expressing SYTL5-EGFP co-stained with 50 nM MitoTracker red. SYTL5-EGFP expression was induced for 24 hr using 100 ng/ml doxycycline. MitoTracker Red was added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( B ) Time-lapse video frames tracking SYTL5 vesicle movement along filaments positive for MitoTracker red (arrows). SYTL5-EGFP expression was induced for 24 hr using 100 ng/ml doxycycline. Scale bars: 3 μm. ( C ) Live confocal microscopy imaging of U2OS stably expressing SYTL5-EGFP co-stained with 50 nM LysoTracker red. SYTL5-EGFP expression was induced for 24 hr using 100 ng/ml doxycycline. LysoTracker Red was added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( D ) Confocal imaging of U2OS stably expressing SYTL5-EGFP stained for endogenous LAMP1. Nuclei were stained using Hoechst. Scale bars: 10 μm, 2 μm (insets). ( E ) Overview of domain structure of SYTL5 full length, ∆SHD and ∆C2AB mutants. ( F ) 3xFLAG fusion proteins expressed in U2OS cells (SYTL5-EGFP-3xFLAG, SYTL5 (∆SHD)-EGFP-3xFLAG and SYTL5 (∆C2AB)-EGFP-3xFLAG) were immunoprecipitated and added to membranes spotted with lipids found in cell membranes: LPA (lipoprotein A); LPC (lysophosphatidylcholine); PI (phosphatidylinositol); PI(3)P (phosphatidylinositol 3-phosphate); PI(4)P (phosphatidylinositol 4-phosphate); PI(5)P (phosphatidylinositol 5-phosphate); PE (phosphatidylethanolamine); PC (phosphatidylcholine); S1P (sphingosine-1-phosphate); PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate); PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate); PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate); PI(3,4,5)P3 (phosphatidylinositol 3,4,5-triphosphate); PA (phosphatidic acid); PS (phosphatidylserine); TAG (triglyceride); DAG (diglyceride); PG (phosphatidylglycerol); CL (cardiolipin); Cholesterol; SM (sphingomyelin); Sulfatide. ( G ) Live confocal microscopy imaging of U2OS stably expressing SYTL5 (∆SHD)-EGFP-3xFLAG co-stained with 50 nM MitoTracker red added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( H ) Live confocal microscopy imaging of U2OS stably expressing SYTL5 (∆C2AB)-EGFP-3xFLAG co-stained with 50 nM MitoTracker red added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). Figure 1—source data 1. Original image files for . Figure 1—source data 2. Original uncropped membranes for . Figure 1—source data 3. Uncropped membranes with the relevant spots clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Confocal Microscopy, Imaging, Stable Transfection, Expressing, Staining, Immunoprecipitation

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A–F ) SYTL5-EGFP expression was induced for 24 hr with 100 ng/ml doxycycline in U2OS cells with stable inducible expression of SYTL5-EGFP and constitutive expression of mScarlet-RAB4 ( A ), mScarlet-RAB11 ( B ), mScarlet-RAB5 ( C ), mScarlet-RAB7 ( D ), mScarlet-RAB9 ( E ), or mScarlet-RAB6 ( F ) followed by live confocal microscopy imaging analysis. Scale bars: 10 μm, 2 μm (insets). Figure 1—figure supplement 1—source data 1. Original image files for .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Expressing, Confocal Microscopy, Imaging

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5-EGFP expression was induced for 24 hr with 100 ng/ml doxycycline in U2OS cells with stable inducible expression of SYTL5-EGFP and constitutive expression of mScarlet-RAB27A. Cells were co-stained with MitoTracker DR for 30 min before live imaging. Arrows indicate mitochondrion filaments. Scale bars: 10 μm, 2 μm (insets). ( B ) CLEM analysis of the cells described in A. Before imaging, cells growing in monolayer were fixed in warm (≈37 °C) 3.7% paraformaldehyde in 0.2 M HEPES (pH 7). After fixation, cells were imaged using a confocal microscope to acquire Z-stacks of optical sections and DIC images to locate the cells of interest. The cells were finally fixed using 2% glutaraldehyde in 0.2 M HEPES (pH 7.4) for 120 min before sample preparation for TEM. Arrows in panel 1 indicate mitochondrion filaments. Scale bars: 15 μm (left), 5 μm (middle) and 2 μm (right). ( C ) U2OS dKO cells were rescued with mScarlet-RAB27A and co-stained with 50 nM MitoTracker green for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( D ) U2OS dKO cells were rescued with SYTL5-EGFP and co-stained with 50 nM MitoTracker red for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( E ) Live confocal microscopy imaging of U2OS dKO cells rescued with SYTL5-EGFP and mScarlet-RAB27A (upper panel); SYTL5-EGFP and mScarlet-RAB27A-T23N (middle panel) or SYTL5-EGFP and mScarlet-RAB27A-Q78L (lower panel). All cells were co-stained with 50 nM MitoTracker DR for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( F ) Lysates from U2OS cells stably expressing EGFP (control) and U2OS dKO cells expressing SYTL5-EGFP and/or mScarlet-RAB27A (wild-type, T23N, or Q78L mutants) were immunoprecipitated using GFP-Trap beads and analysed by western blot using RAB27A and EGFP antibodies. Figure 2—source data 1. Original image files for . Figure 2—source data 2. Original uncropped blots for . Figure 2—source data 3. Uncropped blots with the relevant bands clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Expressing, Staining, Imaging, Microscopy, Sample Prep, Confocal Microscopy, Stable Transfection, Control, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) Live confocal microscopy imaging of U2OS constitutively expressing mScarlet-RAB27A co-stained with MitoTracker green added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( B ) Total cell lysate (TCL), cytosol, and a crude mitochondria fraction from U2OS control cells and cells with stable expression of mScarlet-RAB27A or SYTL5-EGFP were analysed by western blotting for EGFP, RAB27A, tubulin, TIM23, and COXIV. ( C ) Live confocal microscopy imaging of HeLa constitutively expressing SYTL5-EGFP co-stained with MitoTracker Red added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( D ) Live confocal microscopy imaging of HeLa constitutively expressing mScarlet-RAB27A co-stained with MitoTracker green added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). Figure 2—figure supplement 1—source data 1. Original image files for , C, D. Figure 2—figure supplement 1—source data 2. Original uncropped blots for . Figure 2—figure supplement 1—source data 3. Uncropped blots with the relevant bands clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Confocal Microscopy, Imaging, Expressing, Staining, Control, Western Blot

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5 interactome. SYTL5-EGFP expression in U2OS cells was induced for 24 hr using 100 ng/ml doxycycline, followed by immunoprecipitation of SYTL5-EGFP using GFP-Trap beads and identification of co-purified proteins by MS analysis. The list of co-purified proteins was compared with that obtained from cells expressing EGFP, and only significant (p<0.05) SYTL5-EGFP-specific protein hits are highlighted in colour. Protein hits falling into at least one of the biological processes: autophagy, regulation of mitochondrion organisation, protein insertion into mitochondrial membrane, or cellular response to oxidative stress are colour coded according to plot legend. All other significant hits are indicated in blue. Non-significant (p>0.05) hits are indicated in grey. Data are from three biological replicates. ( B ) GO cellular compartment term enrichment of significant protein hits co-purified with SYTL5-EGFP. Corresponding enrichment false discovery rate (FDR) value is represented inside each bar and bars are ordered from smallest to largest FDR (q-value) from top to bottom. ( C ) GO biological processes term enrichment of significant protein hits co-purified with SYTL5-EGFP. Corresponding enrichment FDR value is represented inside each bar and bars are ordered from smallest to largest FDR (q-value) from top to bottom. Proteins corresponding to each biological process category are listed in the table to the right.

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Expressing, Immunoprecipitation, Purification, Membrane

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were untreated (upper panel) or treated with 1 mM DFP (2nd panel), hypoxia (1% O 2 ) (3rd panel), or 1 mM DMOG (4th panel) for 24 hr. All cells were stained with 50 nM MitoTracker DR 30 min prior to live confocal microscopy imaging. Scale bars: 10 μm, 2 μm (insets). ( B ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated or not with 1 mM DFP for 24 hr. Cells were stained with 50 nM MitoTracker DR prior to fixation. Fixed cells were stained with a LAMP1 antibody and imaged by confocal microscopy. Scale bars: 10 μm, 2 μm (insets). Arrowheads point to SYTL5/RAB27A-positive structures. ( C ) U2OS cells expressing pSu9-Halo-mGFP were transfected with 20 nM control siRNA, SYTL5 siRNA #1 or RAB27A siRNA #1 for 48 hr before 20 min incubation with the TMR-conjugated Halo ligand followed by 24 hr incubation with 1 mM DFP or 1 mM DMOG. Cell lysates were analysed by western blot for the Halo tag. Actin was used as a loading control. ( D ) Quantification of data in C. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. *=p < 0.05, **=p < 0.01, ***=p < 0.001. Figure 4—source data 1. Original image files for . Figure 4—source data 2. Original image files for . Figure 4—source data 3. Original uncropped blots for . Figure 4—source data 4. Uncropped blots with the relevant bands clearly labelled for and quantification of blots in 2D.

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Staining, Confocal Microscopy, Imaging, Expressing, Transfection, Control, Incubation, Western Blot, Standard Deviation, Comparison

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 mM DFP for 24 hr. Fixed cells were stained with a LAMP1 antibody in addition to either p62 (upper panel) or LC3 (lower panels) antibodies and imaged by confocal microscopy. Arrowheads point to SYTL5/RAB27A-positive structures. Scale bars: 10 μm, 2 μm (insets). ( B ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 mM DFP for 24 hr and 100 nM BafA1 for the final 4 hr. All cells were stained with 50 nM MitoTracker DR 30 min prior to fixing. Fixed cells were stained with either LAMP1 (upper panel), p62 (middle panel), or LC3 (lower panel) antibody and imaged by confocal microscopy. Arrowheads point to SYTL5/RAB27A-positive structures. Scale bars: 10 μm, 2 μm (insets). ( C ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 μM MRT68921 for 2 hr (upper panel) or 5 μM Vps34-IN1 for 2 hr (lower panel). All cells were stained with 50 nM MitoTracker DR 30 min prior to live imaging confocal microscopy. Scale bars: 10 μm, 2 μm (insets). Figure 4—figure supplement 1—source data 1. Original image files for . Figure 4—figure supplement 1—source data 2. Original image files for . Figure 4—figure supplement 1—source data 3. Original image files for .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Staining, Confocal Microscopy, Imaging

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) U2OS cells were transfected with 20 nM control siRNA, SYTL5 siRNA #1 or RAB27A siRNA #1 for 48 hr followed by 24 hr incubation with 1 mM DFP or 1 mM DMOG. Cell lysates were analysed by western blot for BNIP3L. Actin was used as a loading control. Error bars represent the mean with standard deviation between replicates (n=3). ( B ) U2OS cells were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 72 hr. Transfection efficiency was analysed by qPCR where the relative mRNA expression of SYTL5 was normalised to the expression levels of TBP and siControl. Error bars represent the standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Dunnett multiple comparison test, ***=p < 0.001. ( C ) U2OS cells expressing the mitochondrial matrix reporter NIPSNAP1 aa1-53 -EGFP-mCherry (referred to as IMLS cells) were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 hr before 24 hr incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 hr. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed, representing mitochondria delivered to lysosomes as the EGFP signal is quenched in the acidic lysosome) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the control siRNA, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. ( D ) U2OS IMLS cells expressing PARKIN were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 hr before a 16 hr incubation with 20 μM CCCP to induce mitophagy, with treatment with 100 nM BafA1 or not for the last 2 hr. The area of red-only puncta per cell (>1000 cells analysed) was quantified using Cell Profiler from widefield images obtained using a high-content imaging microscope. The obtained results were normalised to the control siRNA, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ( E ) U2OS IMLS cells were transfected with 20 nM control siRNA or RAB27A siRNA oligos for 48 hr before 24 hr incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 hr. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the siControl, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. ( F ) WT (control) or SYTL5 KO U2OS cells were starved in EBSS for 4 hr or incubated with complete medium in the presence or absence of 100 nM BafA1 the last 2 hr. Cell lysates were harvested and analysed by western blot for LC3B and actin proteins. LC3B-II band intensity was quantified relative to actin and normalised to control. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Bonferroni multiple comparison test. Figure 4—figure supplement 2—source data 1. Original uncropped blots for . Figure 4—figure supplement 2—source data 2. Uncropped blots with the relevant bands clearly labelled for . Figure 4—figure supplement 2—source data 3. Values plotted in .

Article Snippet: Antibody , Mouse monoclonal EGFP , Clontech , #632569 , 1:1000.

Techniques: Transfection, Control, Incubation, Western Blot, Standard Deviation, Expressing, Comparison, Staining, Imaging, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Directed evolution of novel AAV variants using the MCMS library for enhanced CNS tropism and reduced liver targeting in mice

doi: 10.1016/j.omtm.2025.101522

Figure Lengend Snippet: Individual characterization of BRC variants in C57BL/6J mice (A) Quantification of EGFP mRNA expression in the brain, spinal cord, and liver 3 weeks after i.v. injection of each capsid in C57BL/6J mice (5E11 vg per mouse, N = 3–4), with expression values normalized to AAV9. The dashed gray line represented the AAV9-CAG-EGFP expression normalized to 1. (B) Quantification of vector genomes in the brain, spinal cord, and liver of C57BL/6J mice, shown as EGFP copy numbers per microgram of total DNA. (C and D) Representative images of EGFP fluorescence in the whole brain and liver. Scale bar: 3 mm (brain) and 5 mm (liver).

Article Snippet: For immunohistochemistry, sections were incubated with a primary monoclonal mouse anti-EGFP antibody (Thermo Fisher Scientific, MA1-952) overnight at 4°C.

Techniques: Expressing, Injection, Plasmid Preparation, Fluorescence

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Directed evolution of novel AAV variants using the MCMS library for enhanced CNS tropism and reduced liver targeting in mice

doi: 10.1016/j.omtm.2025.101522

Figure Lengend Snippet: Characterization of BRC05 and BRC06 variants in BALB/c mice (A) Quantification of EGFP mRNA expression in the brain, spinal cord, and liver 3 weeks after i.v. injection of BRC05 or BRC06 variant in BALB/c mice (5E11 vg per mouse, N = 3–4), with expression values normalized to AAV9. The dashed gray line represented the AAV9-CAG-EGFP expression normalized to 1. (B) Quantification of vector genomes in the brain, spinal cord, and liver of BALB/c mice, shown as EGFP copy numbers per microgram of total DNA. (C) Immunostaining of the whole brain, spinal cord, and liver. Scale bars: 3 mm (brain), 500 μm (spinal cord), and 2 mm (liver).

Article Snippet: For immunohistochemistry, sections were incubated with a primary monoclonal mouse anti-EGFP antibody (Thermo Fisher Scientific, MA1-952) overnight at 4°C.

Techniques: Expressing, Injection, Variant Assay, Plasmid Preparation, Immunostaining

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a Pan-cancer analysis of TRIM15 expression. mRNA expression levels (log2(TPM + 1)) of TRIM15 across different tumor types from The Cancer Genome Atlas (TCGA) were shown as box plots, with the midline representing the mean value. b HEK293T cells transfected with the indicated plasmids were subjected to Co-IP. IP samples and whole cell lysates (WCL) were analyzed by Western blot. c Co-IP analysis of the interaction between Flag-TRIM15 and endogenous Axin1 in HCT116 cells. IP samples and WCL were analyzed by Western blot. d Cell lysates derived from HCT116 cells were analyzed by co-IP with control IgG or anti-Axin1 antibody. IP samples and WCL were analyzed by Western blot. e Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-TRIM15, both of which were purified from bacteria. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot and/or Ponceau S staining. f Immunofluorescence staining of Flag-TRIM15 and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. g Schematic illustration of full-length and truncated Axin1. h Flag-TRIM15, purified from HEK293T cells, was incubated with immobilized GFP-fused Axin1 or its fragments, all of which were also purified from HEK293T cells. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot. i HEK293T cells transfected with TRIM15-YFP along with either Flag-Axin1 or Flag-Axin1 ΔD plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot.

Article Snippet: Antibodies against β-Tubulin (TA503129), GFP (TA150052), and HSP90 (TA500494) were purchased from Origene.

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Control, Purification, Incubation, Bacteria, SDS Page, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a HEK293T cells transfected with the indicated plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. b Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-fused TRIM15 proteins, all of which were purified from bacteria. The pulldown samples and input were analyzed by Western blot and/or Ponceau S staining. c Purified Flag-TRIM15 or Flag-TRIM15 ΔRING proteins were incubated with immobilized GFP-fused Axin1 or E fragment. The pulldown and input samples were analyzed by Western blot. d Immunofluorescence staining of Flag-TRIM15 ΔRING and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. e Purified Flag-TRIM15 or its deletion proteins were incubated with immobilized GFP-Axin1. The pulldown samples and input were analyzed by Western blot. f Purified Flag-Axin1 was incubated with immobilized GST, GST-fused TRIM15, or its deletion proteins. The pulldown and input samples were analyzed by Western blot and/or Ponceau S staining. g Cell lysates from HEK293T cells transfected with the indicated plasmids were boiled in SDS-containing buffer, diluted, and d-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. h HEK293T cells transfected with Flag-Axin1 with or without GFP-TRIM15. Protein stability was examined by collecting samples at the indicated time after CHX (100 μg/mL) treatment.

Article Snippet: Antibodies against β-Tubulin (TA503129), GFP (TA150052), and HSP90 (TA500494) were purchased from Origene.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Purification, Incubation, Bacteria, Staining, Immunofluorescence