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

Summary

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

Abstract

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 "3D Objects Counter" 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.

Introduction

Macroautophagy (commonly referred to as autophagy) is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles^1,2. 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 degradation^1,4.

Autophagosomes are decorated by the aut....

Protocol

1. Cell preparation

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

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
10x Phosphate-Buffered Saline (PBS)	Corning	46-013-CM
12 mm round coverslips	HDA	CBR_OBJ_6467
24 Well- Cell Culture Plate	Sorfa	220300
Absolute ethanol	Biopack	2207.10.00
Alexa Fluor 594 Donkey anti-rabbit IgG (H+L)	Invitrogen	R37119
Confocal Laser Scanning Microscope	Zeiss	LSM 800
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)	Invitrogen	62248
Dexamethasone	Sigma Aldrich	D4902
DMEN	Sartorius	01-052-1A
Fetal Bovine Serum	NATOCOR	Lintc-634
Gemcitabina	Eli Lilly	VL7502
LC3B (D11) XP Rabbit mAb	Cell Signaling Technology	3868S
Methanol	Anedra	6197
Parafilm "M" (Laboratory Sealing Film)	Bemis/Curwood	PM-996
Pen-Strep Solution	Sartorius	03-031-1B
PP242	Santa Cruz Biotechnology	SC-301606
Trypsin EDTA	Gibco	11570626