This work was supported by funding to Y.T. from Japan Society for the Promotion of Science KAKENHI [Grant Number 23K06837] (Tokyo, Japan) and Takeda Science Foundation (Osaka, Japan). The authors appreciate Dr. David C. Rubinsztein (Cambridge Institute for Medical Research, Cambridge, UK) for supplying HeLa cells.

1. Preparation of the cell, P1, and P2 EV fraction from the cultured medium of HeLa cells
<ol>
	<li>Preparation of the P1 and P2 EV fraction from the cultured medium of HeLa cells
	<ol>
		<li>Day 1
		<ol>
			<li>Count the cells using a cell counter and seed 1 &#215; 106 cells on a 6 cm plate containing a culture medium composed of DMEM High Glucose, 10% FBS, and 1% penicillin/streptomycin. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 2 (optional)
		<ol>
			<li>Prepare 20 &#181;M siRNA solution.</li>
			<li>Mix 100 &#181;L of reduced serum medium and 3 &#181;L of siRNA in a low-retention tube to prepare solution A.</li>
			<li>Mix 100 &#181;L of reduced serum medium and 5 &#181;L of transfection reagent in a low-retention tube to prepare solution B.</li>
			<li>Mix solution A and solution B, then incubate the mixture at room temperature (RT) for 5 min.</li>
			<li>Add the mixture of solutions A and B to the medium used for culturing HeLa cells. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 3
		<ol>
			<li>Wash the cells with PBS and expose them to 500 &#181;L of 0.25% trypsin and 1 mM EDTA solution at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
			<li>Add 2.5 mL of the culture medium to the cells, and use a Pasteur transfer pipette with a 200 &#181;L pipette tip attached to prepare a single-cell suspension.</li>
			<li>Count the cells using a cell counter, and seed 1 &#215; 106 cells on a 10 cm plate. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h. 
			NOTE: It is necessary to prepare at least 1 &#215; 106 cells to collect EVs from the culture medium required for immunoblotting analysis.</li>
		</ol>
		</li>
		<li>Day 4
		<ol>
			<li>Prepare culture medium (see step 1.1.1.1) containing either vehicle (DMSO) or 100 nM Bafilomycin A1 (Baf).</li>
			<li>Expose the cells to the medium containing either vehicle or 100 nM Baf and incubate them at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 5
		<ol>
			<li>Collect all the culture medium from the 10 cm plate, transfer it into a 15 mL tube, and centrifuge it at 3,000 &#215; g for 10 min at 4 &#176;C to remove cell debris. 
			NOTE: The remaining cells in the 10 cm plate will be used in section 1.2.1.</li>
			<li>For the isolation of the P1 EV fraction, transfer the supernatant after centrifugation at 3,000 &#215; g into a centrifugation tube, and then centrifuge it at 20,000 &#215; g at 4 &#176;C for 1 h.</li>
			<li>For the isolation of the P2 EV fraction, transfer the supernatant after centrifugation at 20,000 &#215; g into an ultracentrifuge tube, and then centrifuge it at 110,000 &#215; g at 4 &#176;C for 1 h.</li>
			<li>After wiping off the excess moisture inside the tube with a laboratory wipe, resuspend the remaining pellets in the centrifugation tube in 50 &#181;L of 2x sample buffer [2.5% SDS, 125mM Tris-HCl buffer (pH 6.8), 30% glycerol, 10% 2-mercaptoethanol, 0.4% Bromophenol Blue] to prepare the P1 EV fraction.</li>
			<li>Remove the supernatant by decanting, wipe off the excess moisture inside the tube with a laboratory wipe, and then resuspend the remaining pellets in the ultracentrifugation tube in 50 &#181;L of 2x sample buffer to prepare the P2 EV fraction.</li>
			<li>Incubate the P1 and P2 EV fractions at 100 &#176;C on a heat block for 10 min. 
			NOTE: Do not shake the tubes after centrifugation at 20,000 &#215; g and 110,000 &#215; g to avoid accidentally discarding the remaining pellets. After tilting the tube to transfer the supernatant, maintain the tube&#39;s position to prevent the pellet from being immersed in the remaining supernatant again.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of the cell fraction from cultured HeLa cells
	<ol>
		<li>Day 5
		<ol>
			<li>After transfer of the culture medium from the 10 cm plate into a 15 mL tube, wash the cells in the 10 cm dish with PBS and expose them to 2 mL of 0.25% trypsin and 1 mM EDTA solution at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
			<li>Add 8 mL of growth medium to the cells, then use a Pasteur transfer pipette with a 200 &#181;L pipette tip to pipette the cells and prepare a single-cell suspension.</li>
			<li>Transfer the cell suspension into a 15 mL tube and centrifuge it at 1,000 &#215; g for 10 min at 4 &#176;C.</li>
			<li>Remove the supernatant by an aspirator, add PBS to the cell pellet, and then centrifuge at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
			<li>Remove the PBS using an aspirator and sonicate the cell pellet in 1 mL of A68 buffer [10 mM Tris-HCl buffer, pH 7.5, 0.8 M NaCl, 1 mM ethylene glycol bis(&#946;-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)] containing 1% sarkosyl.</li>
			<li>Measure the protein concentration of the cell lysate using a BCA protein assay kit.</li>
			<li>Mix 300 &#181;L of the cell lysate with 100 &#181;L of 4x sample buffer and incubate the mixture at 100 &#176;C on a heat block for 10 min to prepare the cell fraction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Immunoblotting analysis
<ol>
	<li>Separate equivalent amounts of proteins in the samples by SDS-PAGE18.</li>
	<li>Transfer the separated proteins onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>After transfer, block the membranes with the blocking agent for 20 min.</li>
	<li>Incubate the blocked membrane overnight with the indicated primary antibody in Tris buffer containing 10% (v/v) calf serum at RT.</li>
	<li>Wash the membrane with Tris buffer for 5 s and then incubate it with a biotin-conjugated secondary antibody at RT for 2 h.</li>
	<li>Wash the membrane with Tris buffer for 5 s and then incubate it with Tris buffer containing 0.4% (v/v) solutions A and solution B from the kit at RT for 1 h.</li>
	<li>Wash the membrane with PBS, and then incubate it with PBS containing 0.04% (w/v) diaminobenzidine, 0.8% (w/v) NiCl2&#183;6H2O, and 0.3% (v/v) H2O2 for colorimetric detection of bands. 
	NOTE: For the detection of signals, a chemiluminescence method is also acceptable.</li>
	<li>Digitize the image of the membrane by a scanner and subject it to densitometry analysis using Fiji.
	<ol>
		<li>Open the digitized image using Fiji, and convert the RGB color image to an 8-bit image by clicking Image | Type | 8-bit.</li>
		<li>Click on the rectangle tool and adjust the size of the rectangle by dragging it to surround the bands. Identify the band to be analyzed by clicking Analyze | Gels | Select First lane.</li>
		<li>Position the rectangle on the second and the subsequent bands, and identify the bands for analysis by clicking Analyze | Gels | Select Next Lane.</li>
		<li>After recognizing the final band, measure the densitometry across the rectangle by clicking Analyze | Gels | Plot Lanes.</li>
		<li>Surround the signal area of the band with a straight line, click the wand tool, and click in the area to calculate the signal intensity of the band.</li>
	</ol>
	</li>
</ol>
3. Analysis of the number of autophagosomes and autolysosomes
<ol>
	<li>Day 1
	<ol>
		<li>Count the number of HeLa cells using a cell counter, and seed 1 &#215; 105 HeLa cells expressing LAMP1-GFP and mCherry-LC3 on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 2 (optional)
	<ol>
		<li>Perform the steps described in section 1.1.2 in 3.5 cm dishes.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Discard the culture medium from the 3.5 cm dishes.</li>
		<li>Wash cells with PBS, expose them to 400 &#181;L of 0.25% trypsin and 1 mM EDTA solution, and incubate at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
		<li>Add 1.6 mL of the growth medium to the cells, and pipette them with a Pasteur transfer pipette to prepare a single-cell suspension.</li>
		<li>Count the number of cells using the cell counter, and seed 1 &#215; 104 cells on an 8-chambered coverslip. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h. 
		NOTE: Two wells of the chambered coverslip are prepared for control and STX6 knockdown cells, respectively, following DMSO or Baf treatment as described in section 3.4.2.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Prepare the growth medium without phenol red (DMEM without Phenol Red, 10% FBS, 1% penicillin/streptomycin), containing either vehicle (DMSO) or 100 nM Baf dissolved in DMSO.</li>
		<li>Replace the culture medium with the medium without phenol red, containing either vehicle or 100 nM Baf, and incubate for 4 h. 
		NOTE: To assess the effect of Baf on autophagosome and autolysosome numbers, prepare vehicle-treated cells as controls.</li>
		<li>Stain the nuclei with 0.33 &#181;g/mL Hoechst 33342 for 30 min before observation.</li>
		<li>Conduct Live cell imaging using a 60x objective lens on a confocal laser microscope and capture ten different frames.
		<ol>
			<li>Open the RGB merged image in Fiji, and split it into the respective red, green, and blue image components by clicking on Image | Color | Split Channels.</li>
			<li>Set a constant threshold for the red and green signals in each image by clicking Image | Adjust | Threshold for each experiment.</li>
			<li>Count the number of areas positive for red or green signals by clicking Analyze | Analyze Particles. Check the Summarize box, then click OK. Record the values from the Count to identify autophagosome- and lysosome-associated vesicles.</li>
			<li>To overlay the green image on the red image, set the transfer mode to AND by clicking Edit | Paste Control. Copy the green image and then paste it onto the red image to merge them, highlighting areas that are positive for both red and green signals.</li>
			<li>To identify autolysosome numbers, count the number of areas positive for both red and green signals by clicking Analyze | Analyze Particles. Check the Summarize box, click OK, and record the values in the Count section to identify vesicles associated with autophagosomes and lysosomes.</li>
		</ol>
		</li>
		<li>Divide the total number of autolysosomes and autophagosomes by the total number of cells to calculate the number of autolysosomes and autophagosomes per cell. 
		NOTE: Count at least 35 cells in each of the three independent experiments.</li>
	</ol>
	</li>
</ol>
4. Analysis for omegasome formation
<ol>
	<li>Day 1
	<ol>
		<li>Count the number of HeLa cells using a cell counter, and seed 1 &#215; 105 cells on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 2 (optional)
	<ol>
		<li>Perform the steps described in section 1.1.2.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Prepare a single-cell suspension as described in steps 3.3.1-3.3.2.</li>
		<li>Count the cells using the cell counter, and seed 1 &#215; 105 cells on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Mix 1 &#181;g of pEGFP-C1-hAtg13, 3 &#181;L of the DNA transfection reagent, and 100 &#181;L of reduced serum medium in a low retention tube. Incubate the mixture for 20 min, then add it to the cultured medium.</li>
		<li>Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Trypsinize the cells as described in steps 3.3.1-3.3.2.</li>
		<li>Add 2.5 mL of the growth medium to the cells. Pipette the mixture using a Pasteur transfer pipette to prepare a single-cell suspension.</li>
		<li>Count the cells using the cell counter, and seed 1 &#215; 104 cells on&#160;an 8-chambered coverslip. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 6
	<ol>
		<li>Follow steps 3.4.1-3.4.4 to conduct live cell imaging of the cells from step 4.5.3 using a confocal laser microscope. Capture ten different frames of images.
		<ol>
			<li>Follow steps 3.4.4.1-3.4.4.3 to split the RGB merged images into the respective red, green, and blue image components, set a constant threshold for the green signals in each image, and determine GFP signal intensity. Record the values from the Total Area section to determine the signal intensity of GFP-ATG13.</li>
			<li>Divide the total signal intensity by the number of cells examined to calculate the signal intensity per cell. 
			NOTE: Count at least 34 cells in each of the three independent experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Assessing LC3-II-Positive EV Release in Autophagy with Impaired Lysosome Fusion

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The authors declare that they have no conflicts of interest with the contents of this article.

The immunoblotting study revealed LC3-II and TSG101 levels in the cellular fraction, the microvesicle-rich P1 EV fraction, and the exosome-rich P2 EV fraction. Live-cell imaging was used to examine autophagosomes, autolysosomes, and omegasomes, providing insight into whether STX6 knockdown influences autophagy. These combined results suggest that STX6 knockdown affects the release of LC3-II positive EVs, potentially linked to dysregulation of the autophagy pathway. A key advantage of this method is its ...

Transactivation response DNA-binding protein 43 (TDP-43) is a widely expressed heterogeneous nuclear ribonucleoprotein involved in regulating exon splicing, gene transcription, and mRNA stability, all vital for cell survival1,2. In neurodegenerative conditions like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a nuclear protein TDP-43 abnormally accumulates in the cytoplasm. This shift results in a loss of TDP-43 function in the nucleus and a toxic gain-of-function in the cytoplasm. Pathological accumulation of TDP-43 begins in specific regions of the brain and spinal cord, spreading through these areas in a prion-like fashion, a process closely associated with disease progression3. However, the exact mechanism by which TDP-43 pathology spreads through the brain and spinal cord remains unknown.
TDP-43 is secreted through extracellular vesicles (EVs), and elevated levels of TDP-43 are detected in the plasma and cerebrospinal fluid (CSF) of ALS and FTLD-TDP patients4,5,6. CSF from patients diagnosed with ALS and FTLD induces intracellular mislocalization and aggregation of endogenous TDP-43 in human glioma cells7. Thus, TDP-43 released extracellularly via EVs may mediate the cell-to-cell spreading of TDP-43 pathology.
Autophagy is a well-preserved cellular degradation mechanism that involves the enclosure of unwanted substances within double-membrane vesicles, known as autophagosomes, which are marked by LC3. These autophagosomes fuse with lysosomal-associated membrane protein 1 (LAMP1)-positive lysosomes to form autolysosomes (LC3+/LAMP1+), leading to the degradation of their contents8. Histological analyses indicate that autophagosomes engulfing inclusions accumulate in the neurons of sporadic ALS patients9. Some causal genes of familial ALS and FTLD-TDP are linked to the regulation of autophagy10,11,12. These findings suggest that autophagy is suppressed in ALS and FTLD-TDP patients.
The previous study indicated that TDP-43 is secreted via EVs positive for the autophagosome marker LC3-II when autophagosome-lysosome fusion is inhibited13. Dysregulation of the autophagy-lysosome pathway might cause not only the intracellular accumulation of TDP-43 but also the extracellular release of TDP-43 via EVs14. However, it remains unknown how LC3-II positive EVs are released and how significant this is in the spreading of TDP-43 pathology.
EVs are classified into large EVs (100 to 1000 nm in diameter), which are produced from cell-surface budding, and small EVs (50 to 150 nm in diameter), which are produced from the budding of endosomal membranes toward the interior of the endosome (known as exosomes) and the Golgi apparatus. To separately collect large and small EVs, we perform sequential centrifugation and collect pellets by centrifugation at 20,000 &#215; g and 110,000 &#215; g, respectively. The P1 EV fraction (20,000 &#215; g pellets) is prepared to collect large EVs, and the P2 EV fraction (110,000 &#215; g pellets) is prepared to collect small EVs15. The methodology to assess LC3-II levels in large and small EVs derived from sequential centrifugation by immunoblotting is stable and reproducible16. In addition to analyzing LC3-II levels in EVs, analyzing autolysosome and omegasome formation enhances understanding of how dysregulation of autophagy leads to the release of LC3-II positive EVs. Here, the present study shows the suppressive role of syntaxin 6 (STX6), a SNARE protein, which promotes the movement of transport vesicles to target membranes17, in the release of LC3-II positive EVs when autophagosome-lysosome fusion is inhibited.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>0.25 % Trypsin EDTA</td>
			<td>Fujifilm Wako</td>
			<td>201-16945</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>150464</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L Pipette Tip</td>
			<td>Nippon Genetics</td>
			<td>FG-301</td>
			<td>pipetting</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Nacalai Tesque</td>
			<td>21417-52</td>
			<td>a material for 
			sample buffer solution</td>
		</tr>
		<tr>
			<td>3,3&#39;-Diaminobenzidine Tetrahydrochloride</td>
			<td>Nacalai Tesque</td>
			<td>11009-41</td>
			<td>a material for DAB solution</td>
		</tr>
		<tr>
			<td>3.5 cm Dish&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Dish&#160;</td>
			<td>TrueLine</td>
			<td>TR4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium Block Thermostatic Baths (dry thermobaths)</td>
			<td>EYELA</td>
			<td>273860</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td>SANSYO</td>
			<td>SAP-102</td>
			<td>inhaling solution</td>
		</tr>
		<tr>
			<td>Avanti JXN-30</td>
			<td>Beckman Coulter</td>
			<td>B34193</td>
			<td></td>
		</tr>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Adipogen</td>
			<td>BVT-0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-conjugated Goat Anti-rabbit IgG Antibody</td>
			<td>Vector Laboratories&#160;</td>
			<td>BA-1000</td>
			<td>2nd antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Biotin-conjugated Horse Antimouse IgG Antibody</td>
			<td>Vector Laboratories&#160;</td>
			<td>BA-2000</td>
			<td>2nd antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Blocking One</td>
			<td>Nacalai Tesque</td>
			<td>03953-95</td>
			<td>a material for immunoblotting</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Nacalai Tesque</td>
			<td>05808-61</td>
			<td>a material for 
			sample buffer solution</td>
		</tr>
		<tr>
			<td>Calf Serum</td>
			<td>cytiva</td>
			<td>SH30073.03</td>
			<td></td>
		</tr>
		<tr>
			<td>CanoScan LiDE 220</td>
			<td>Canon</td>
			<td>CSLIDE220</td>
			<td>Scanner</td>
		</tr>
		<tr>
			<td>Centrifuge 5702 R</td>
			<td>eppendolf</td>
			<td>5703000039</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Slides Dual Chamber</td>
			<td>Bio-Rad</td>
			<td>1450015J</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>Digital Sonifier 450</td>
			<td>BRANSON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>nacalai tasque</td>
			<td>09659-14</td>
			<td>vehicle</td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>Nacalai Tesque</td>
			<td>08458-45</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>DMEM without Phenol Red</td>
			<td>Nacalai Tesque</td>
			<td>08489-45</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dojindo&#160;</td>
			<td>348-01311</td>
			<td>a material for A68 solution</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>version 16.16.27</td>
			<td>satistical analysis</td>
		</tr>
		<tr>
			<td>EZR</td>
			<td>Reference No. 24</td>
			<td>version 1.68</td>
			<td>satistical analysis</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>173012</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>NIH</td>
			<td></td>
			<td>Image analysis tool</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Nacalai Tesque</td>
			<td>09886-05</td>
			<td>a material for 
			sample buffer solution</td>
		</tr>
		<tr>
			<td>Hoehst33342</td>
			<td>Dojindo&#160;</td>
			<td>H342</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fujifilm Wako</td>
			<td>080-0186</td>
			<td>a material for DAB solution</td>
		</tr>
		<tr>
			<td>Kimwipe S-200</td>
			<td>NIPPON PAPER CRECIA</td>
			<td>62011</td>
			<td>cleaning wipe</td>
		</tr>
		<tr>
			<td>Low Retention Tube</td>
			<td>Nippon Genetics</td>
			<td>FG-MCT015CLB</td>
			<td>siRNA and DNA transfection</td>
		</tr>
		<tr>
			<td>LSM780 Confocal Laser Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Mouse Anti-LC3 Antibody</td>
			<td>MBL</td>
			<td>M186-3</td>
			<td>1st antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Nickel(II) Chloride Hexahydrate</td>
			<td>Fujifilm Wako</td>
			<td>149-01041</td>
			<td>a material for DAB solution</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine Sodium Salt</td>
			<td>Nacalai Tesque</td>
			<td>20117-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985-070</td>
			<td>siRNA and DNA transfection</td>
		</tr>
		<tr>
			<td>pEGFP-C1-hAtg13</td>
			<td>Addgene</td>
			<td>22875</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Nacalai Tesque</td>
			<td>26253-84</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-PCNA Antibody</td>
			<td>BioAcademia</td>
			<td>70-080</td>
			<td>1st antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-syntaxin 6 Antibody</td>
			<td>ProteinTech</td>
			<td>10841-1-AP</td>
			<td>1st antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-TSG101 Antibody</td>
			<td>ProteinTech</td>
			<td>28283-1-AP</td>
			<td>1st antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-ULK1 Antibody</td>
			<td>ProteinTech</td>
			<td>20986-1-AP</td>
			<td>1st antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Polyvinylidene Difluoride Membrane</td>
			<td>Milliore</td>
			<td>IPVH00010</td>
			<td>a material for immunoblotting</td>
		</tr>
		<tr>
			<td>R</td>
			<td>R development core team</td>
			<td>version 4.4.1</td>
			<td>satistical analysis</td>
		</tr>
		<tr>
			<td>RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778</td>
			<td>siRNA transfection reagent</td>
		</tr>
		<tr>
			<td>siRNA STX6</td>
			<td>Thermo Fisher Scientific</td>
			<td>HSS115604</td>
			<td>siRNA for transfection</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Nacalai Tesque</td>
			<td>31320-05</td>
			<td>a material for Tris 
			buffer and A68 solution</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fujifilm Wako</td>
			<td>192-13981</td>
			<td>a material for 
			sample buffer solution</td>
		</tr>
		<tr>
			<td>SPARK Microplate Reader</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth RNAi Negative Control Duplexes, Med GC</td>
			<td>Thermo Fisher Scientific</td>
			<td>12935300</td>
			<td>siRNA transfection</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fujifilm Wako</td>
			<td>193-00025</td>
			<td>a material for A68 solution</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter with Thermal Printer</td>
			<td>Bio-Rad</td>
			<td>1450109J1</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>Thermobath</td>
			<td>TOKYO RIKAKIKAI</td>
			<td>MG-3100</td>
			<td>incubation</td>
		</tr>
		<tr>
			<td>TransIT-293</td>
			<td>Mirus Bio</td>
			<td>MIR 2700</td>
			<td>DNA transfection reagent</td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Mirus Bio</td>
			<td>MIR2300</td>
			<td>DNA transfection reagent</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Nacalai Tesque</td>
			<td>35406-75</td>
			<td>a material for Tris buffer, sample buffer and A68 solution</td>
		</tr>
		<tr>
			<td>Trypan Blue Dye 0.40%</td>
			<td>Bio-Rad</td>
			<td>1450021</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>Ultra-Clear Open-Top Tube, 16 x 96mm&#160;</td>
			<td>Beckman Coulter</td>
			<td>361706</td>
			<td>collecting for the P1 EV fraction</td>
		</tr>
		<tr>
			<td>Ultra-Clear Tube, 14 x 89mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td>collecting for the P2 EV fraction</td>
		</tr>
		<tr>
			<td>Vectastain ABC Standard Kit</td>
			<td>Vector Laboratories&#160;</td>
			<td>PK-4000</td>
			<td>immunoblotting</td>
		</tr>
		<tr>
			<td>Wash Bottle</td>
			<td>As One</td>
			<td>1-4640-02</td>
			<td>washing membrane</td>
		</tr>
		<tr>
			<td>&#956;-Slide 8 Well High</td>
			<td>ibidi</td>
			<td>80806</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of lc3-ii release via extracellular vesicles in relation to the accumulation of intracellular lc3-positive vesicles

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.
To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

As shown in previous studies, Baf treatment increased the levels of TSG101 (P1: P &#60; 0.01, P2: P = 0.012, as determined by two-way ANOVA in EZR19) and LC3-II (P1: P &#60; 0.01, P2: P &#60; 0.01, as determined by two-way ANOVA in EZR19) in the EV-rich fraction. Notably, Baf treatment also increased the levels of STX6 (P1: P &#60; 0.01, P2: P &#60; 0.01, as determined by two-way ANOVA in EZR19) in the EV fraction (Figure 1A</str...

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf ...

evaluation-lc3-ii-release-via-extracellular-vesicles-relation-to

Research

JoVE Journal

Biochemistry

467 Views.  Okayama University of Science. Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

Video: Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of &lt;200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...

Isolating Small Extracellular Vesicles (sEVs) by Differential Ultracentrifugation

Isolating Small Extracellular Vesicles (SEVs) by Differential Ultracentrifugation  

Extraction of Peptides from Isolated Small Extracellular Vesicles (sEVs)

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...