Name: evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence
Uploaded: 2023-04-28T13:40:00
Description: Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

This work was supported by grants from the University of Buenos Aires (UBACyT 2018-2020 20020170100082BA), the National Council for Scientific Research and Technology (CONICET) (PIP 2021-2023 GI&#8722; 11220200101549CO; and PUE 22920170100033) and the National Agency for Scientific and Technological Promotion (PICT 2019-01664).

1. Cell preparation
<ol>
	<li>Soak 12 mm round coverslips in absolute ethanol, and place them vertically in the wells of a 24-well plate.</li>
	<li>Remove the cover, and expose the multi-well plate to ultraviolet radiation for 15 min.</li>
	<li>Position the coverslips horizontally, and wash them with Dulbecco&#39;s Modified Eagle Medium (DMEM).</li>
	<li>Seed a low passage number of pancreatic cells. The amount should be adjusted to obtain 50%-75% confluency on the day of fixation16</...

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+steps+in+mammalian+autophagosome+biogenesis.">Initial steps in mammalian autophagosome biogenesis.</a> Frontiers in Cell and Developmental Biology. 6, 146 (2018).</li><li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+definitions+of+autophagy+and+related+processes.">Molecular definitions of autophagy and related processes.</a> EMBO Journal. 36 (13), 1811-1836 (2017).</li><li>Kitada, M., Koya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+metabolic+disease+and+ageing.">Autophagy in metabolic disease and ageing.</a> Nature Reviews Endocrinology. 17 (11), 647-661 (2021).</li><li>Ktistakis, N. T., Tooze, S. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+Principles+and+modern+practices.">Confocal microscopy: Principles and modern practices.</a> Current Protocols in Cytometry. 92 (1), 68 (2020).</li><li>Grasso, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+novel+selective+autophagy+pathway+mediated+by+VMP1-USP9x-p62,+prevents+pancreatic+cell+death.">a novel selective autophagy pathway mediated by VMP1-USP9x-p62, prevents pancreatic cell death.</a> Journal of Biological Chemistry. 286 (10), 8308-8324 (2011).</li><li>Ropolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatitis-induced+vacuole+membrane+protein+1+triggers+autophagy+in+mammalian+cells.">The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells.</a> Journal of Biological Chemistry. 282 (51), 37124-37133 (2007).</li><li>Karanasios, E., Stapleton, E., Walker, S. A., Manifava, M., Ktistakis, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+early+autophagy+events:+Omegasomes+and+beyond.">Live cell imaging of early autophagy events: Omegasomes and beyond.</a> Journal of Visualized Experiments. (77), e50484 (2013).</li><li>Kuma, A., Matsui, M., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+an+autophagosome+marker,+can+be+incorporated+into+protein+aggregates+independent+of+autophagy:+caution+in+the+interpretation+of+LC3+localization.">LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization.</a> Autophagy. 3 (4), 323-328 (2007).</li><li>Zhang, Z., Singh, R., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+of+autophagy+in+mammalian+cells.">Methods for the detection of autophagy in mammalian cells.</a> Current Protocols in Toxicology. 69, 1-26 (2016).</li><li>Yoshii, S. R., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+measuring+autophagy.">Monitoring and measuring autophagy.</a> International Journal of Molecular Sciences. 18 (9), 1865 (2017).</li><li>Betriu, N., Andreeva, A., Semino, C. 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The method described in this protocol allows for visualizing the endogenous LC3 distribution in the cell and quantifying the autophagic levels under different conditions. Another similar method used to analyze the LC3 distribution and determine autophagy activation involves fluorescence-labeled LC3 transfection (such as RFP-LC3)19. RFP-LC3 transfection has the advantages of not needing fixation (which allows for applying this method in live cell imaging20), being cheaper, a...

Macroautophagy (commonly referred to as autophagy) is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles1,2. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis3. During autophagy, a double membrane vesicle is formed: the autophagosome. The autophagosome contains the cargo molecules and drives them to the lysosome for degradation1,4. 
Autophagosomes are decorated by the autophagic protein microtubule-associated protein light chain 3 (LC3)5. When autophagy is not induced, LC3 is diffused in the cytoplasm and nucleus in the LC3-I conformation. On the other hand, when autophagy is induced, LC3 is conjugated with a phosphatidylethanolamine in the membrane of the autophagic structures6. This new LC3 conformation is known as LC3-II1. The LC3 conformation shift causes changes in its cellular localization and its dodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration, which can be detected by techniques such as immunofluorescence and western blot5,7. In this way, LC3 conjugation is a key event in the autophagic process that can be used to measure autophagic activity.
The pancreatic acinar cell is a highly differentiated cell that, under healthy conditions, has a low rate of autophagy. However, in different physiological conditions or under pharmacological stimulation, they can activate autophagy. Therefore, the determination of autophagic levels in this cell line is useful for studying the potential direct or indirect effects of different pharmacological or biological agents on autophagy8,9.
Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer, given its late diagnosis and its high chemotherapy resistance10. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy-related proteins11. Pancreatic cancer cells may adjust their autophagy levels in response to unfavorable conditions like hypoxia, nutrient deprivation, or chemotherapy-induced damage11. Hence, analyzing the autophagy levels in pancreatic cancer cells can help understand how they adapt to varying environments and evaluate the effectiveness of autophagy-modulating treatments.
This study shows a method to perform LC3 immunofluorescence in two distinct pancreatic cellular models. The first model, PANC-1 cells, served as a model for pancreatic ductal adenocarcinoma. These cells were treated with gemcitabine, a chemotherapy agent that has previously been shown to induce autophagy, specifically in pancreatic cancer cells carrying the oncogenic Kirsten rat sarcoma virus gene (KRAS)12,13. The second model, AR42J cells, served as a more physiological model of exocrine pancreatic cells. These cells were differentiated with dexamethasone to become more similar to acinar pancreatic cells14. In these cells, autophagy was pharmacologically induced through the use of PP242, which is a potent mTOR inhibitor15. In this study, we demonstrate the applicability of the protocol described with two different pancreatic models and its ability to discriminate between states of low and high autophagy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm round coverslips</td>
			<td>HDA</td>
			<td>CBR_OBJ_6467</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well- Cell Culture Plate</td>
			<td>Sorfa</td>
			<td>220300</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Biopack</td>
			<td>2207.10.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>R37119</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEN</td>
			<td>Sartorius</td>
			<td>01-052-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>NATOCOR&#160;</td>
			<td>Lintc-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemcitabina</td>
			<td>Eli Lilly</td>
			<td>VL7502</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3B (D11) XP Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>3868S</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Anedra</td>
			<td>6197</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34; (Laboratory Sealing Film)</td>
			<td>Bemis/Curwood</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep Solution</td>
			<td>Sartorius</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PP242</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-301606</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco</td>
			<td>11570626</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins.
Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the &#34;3D Objects Counter&#34; tool.
The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

This protocol performs immunofluorescence of LC3 in pancreatic cell lines to determine the autophagy levels in different conditions. The outcome of this experiment was the obtention of cellular images from the red and blue channels, corresponding to LC3 and DAPI. The LC3 images indicate the cellular distribution of this protein, whereas the DAPI shows the nuclear localization. Figure 2A shows a representative image of the immunofluorescence of LC3 and its merge with DAPI staining in PANC-1 c...

Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

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