Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 colocalized with the virus, and both colocalized with all endosomes tested. ( A ) H1299-ACE2 cells were transduced with S-EGFP pseudovirus and fixed by paraformaldehyde at 1 h post-transduction. Endogenous STX6 was stained by antibody. The white frames indicated the colocalization of STX6 with S-EGFP. ( B ) H1299-ACE2 cells were transfected with mCherry-STX6 plasmid and then transduced with S-EGFP pseudovirus. One hour post-infection, cells were fixed, and the endogenous EEA1, Rab5, and Rab7 were stained by antibodies. The LAMP1-Flag plasmid was first transfected together with mCherry-STX6 plasmid for LAMP1-Flag staining. White frames indicate colocalization of S-EGFP, mCherry-STX6, and different markers. ( C ) Mock and S-EGFP pseudovirus transduced H1299-ACE2 cells fixed at 1 h post-infection and stained with STX6 and EEA1, Rab5, Rab7, or Flag antibodies. ( D–G ) The subcellular distribution dynamics of endogenous STX6 in Mock and S-EGFP pseudovirus transduced H1299-ACE2 cells. ( D ) The Manders’ colocalization coefficients (MCCs) represent the proportion of STX6 colocalized with different endosome markers in each cell before and after pseudovirus transduction. ( E ) The number of STX6 + marker + puncta in ( D ) was counted. ( F ) The MCCs represent the proportion of marker colocalized with STX6 in each cell before and after pseudovirus transduction. ( G ) The MCCs represent the proportion of STX6 in marker + PP + puncta or in marker + PP − puncta in each cell post-S-EGFP pseudovirus transduction. ( H ) Live cell fluorescence imaging shows the colocalization process of S-EGFP pseudovirus and mCherry-STX6. ( I ) Representative images showing the localization of endogenous STX6 in H1299-ACE2 cells at 24 h post-SARS-CoV-2 infection (MOI = 0.01). The differences between the two groups were determined by two-tailed t tests. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 20. Scale bars, 20 µm.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Virus, Transduction, Staining, Transfection, Plasmid Preparation, Infection, Marker, Fluorescence, Imaging, Two Tailed Test

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 1. Endosomes and lysosomes form giant structures in the periphery of the oocyte plasma membrane. (A) Metaphase II (MII) oocytes were fixed and co-stained with anti-RAB5 and anti-LAMP1 antibodies. The schematic diagram indicates the location of oocyte chromosomes and metaphase plate in the MII oocytes. Arrowheads indicate the positions of oocyte chromosomes. Magnified regions (right) are indicated by yellow boxes. (B) Embryos at

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 2. The giant structures are enlarged in the periphery of the metaphase II (MII) oocyte plasma membrane (PM) during oocyte maturation. Germinal vesicle (GV) oocyte, MII oocyte, early 2-cell (E2C: 24 hr post-fertilization [hpf]), and late 2-cell (L2C: 40 hpf) embryos were fixed and stained with anti-LAMP1 antibody. Reconstituted three-dimensional objects for the LAMP1-positive organelles in 9–10 oocytes for each stage from more than three independent experiments were further analyzed for number, size, and distribution. (A) Averaged total number of LAMP1-positive objects per

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 3. Endosomal-lysosomal organelles form assembly structures, ELYSA. (A) The internal structure of the giant structure in metaphase II (MII) oocytes was analyzed by correlative light and electron microscopy (CLEM) analysis using anti-RAB7 and anti-LAMP1 antibodies. A merged image of immunostaining and electron microscopy (EM) is indicated (left), and a toluidine blue staining image of the adjacent thin section (right) is indicated. Black arrowheads indicate the giant structures positive for RAB7/LAMP1/toluidine blue staining. Note that RAB7/LAMP1 staining is indicated in a

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Electron Microscopy, Immunostaining, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 4. An autophagy regulator LC3 is detected in endosomal-lysosomal organellar assembly (ELYSA). Germinal vesicle (GV) (A) and metaphase II (MII) (B) oocytes were fixed and co-stained with anti-LC3 and anti-LAMP1 antibodies. Magnified regions (right) are indicated by yellow boxes. DNA was stained with Hoechst 33342. Arrowheads indicate the positions of oocyte chromosomes. Maximum intensity projection of confocal images at an axial scan range of 80 µm is shown as Z-projection images.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 5. Enlargement of endosomal-lysosomal organellar assembly (ELYSA) and its redistribution to the cell periphery occur in an actin cytoskeleton- dependent manner. Germinal vesicle (GV) oocytes were incubated for in vitro maturation (IVM) for 18 hr with or without actin polymerization inhibitors (10 μM latrunculin A or cytochalasin B) or a tubulin inhibitor (20 μM nocodazole), then fixed and co-stained with anti-LC3 and anti-LAMP1 antibodies. The reconstituted objects for LAMP1-positive organelles in 10 oocytes for each treatment from more than three independent experiments were

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Incubation, In Vitro, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 6. Assembly of endosomal-lysosomal organellar assembly (ELYSA) during migration and limited interaction with the acidification machinery. (A) Snapshots of a time-lapse observation for EGFP fluorescence of germinal vesicle (GV) oocytes injected with Lamp1-EGFP and V1A-mCherry mRNA are indicated. Maximum intensity projection of confocal images with a height of 40 μm (bottom half of the oocyte) are shown. Magnified perinuclear regions (right) are indicated by yellow boxes. Arrowheads indicate assemblies that adhere to each other in the subsequent frame. (B) Maximum intensity projection of deconvolved confocal images at an axial scan range of 80 µm is shown as Z-projection images. Magnified regions are indicated by yellow boxes (a, b) and the LAMP1-EGFP organelle regions are indicated by yellow dotted line.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Migration, Fluorescence, Injection