Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1

Abstract

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Keywords: Autophagosome; LC3; cancer; flux; lysosome; macroautophagy; neurodegeneration; phagophore; stress; vacuole.

Figure 19.

Assessing autophagy with multispectral imaging cytometry. (A) Bright Detail Intensity (BDI) measures the foreground intensity of bright puncta (that are 3 pixels or less) within the cell image. For each cell, the local background around the spots is removed before intensity calculation. Thus, autophagic cells with puncta have higher BDI values. (B) Media control (untreated wild type), rapamycin-treated wild-type and atg5^−/− MEFs were gated based on BDI. Representative images of cells with high or low BDI values. Scale bar: 10 µm. Images provided by M.L. Albert

Figure 20.

HiBiT-LC3B imitates endogenous LC3B response upon autophagy perturbation. (A) Principle of the Autophagy LC3-HiBiT reporter assay system. (B) HCT116 (parent) and stable HCT116-LC3 HiBiT cell lines were exposed to vehicle control (Veh Ctrl), chloroquine (CQ; 25 μM) or the CSNK1A1/CK1α (casein kinase 1 alpha 1) inhibitor D4476 (10 μM) for 24 h, which increases LC3 abundance [4084]. Cell lysates (30 μg) were resolved in 12% SDS-PAGE gels and transblotted to nitrocellulose membranes. Nano-Glo HiBiT Blotting System (Promega, N2410) was used to detect HiBiT-LC3B (expected molecular size of 55 kDa) using Tris-buffered saline supplemented with 0.1% Tween 20. The HiBiT-LC3B blot was imaged after 20 min incubation with the substrate. Loading control (KIF11/Eg5; Cell Signaling Technology, 4203) and endogenous LC3B (Cell Signaling Technology, 2775) were detected via standard immunoblotting (leftmost lane in immunoblots: protein ladder). Image provided by John J.E. Chua and Jit K. Cheong. (C) U2OS LC3 HiBiT cells treated with autophagy inducers or inhibitors. Effect on autophagic flux was measured in cells incubated in normal or starvation medium (HBSS) alone or containing bafilomycin A₁ (50 nM) or torin1 (25 nM) for 3 h using the HiBiT luminescence assay. Bars are mean ± s.e.m. of triplicate samples. ***P < 0.001 vs. untreated normal media control; t-test. AU, arbitrary units. Image provided by Silvia Vega-Rubin-de-Celis

Figure 21.

Regulation of the SQSTM1 protein during autophagy. (A) The level of SQSTM1 during starvation. Atg5^+/+ and atg5^−/− MEFs were cultured in DMEM without amino acids and serum for the indicated times, and then subjected to immunoblot analysis using anti-SQSTM1antibody (Progen Biotechnik, GP62). This figure was previously published in ref. [30], and is reproduced by permission of Landes Bioscience, copyright 2007. (B) The level of SQSTM1 in the brain of neural-cell specific atg5 knockout mice. Image provided by T. Hara

Figure 22.

S. cerevisiae cells transformed with a plasmid encoding HA-Atg1 were cultured to mid-log phase and shifted to SD-N (minimal medium lacking nitrogen that induces a starvation response). Immunoblotting was done with anti-HA antibody. The upper band corresponds to autophosphorylation of Atg1. This figure was modified from data previously published in ref. [747], and is reproduced by permission of the American Society for Cell Biology, copyright 2011

Figure 23.

Confocal microscopy image of HCT116 cells immunostained with antibody specific to human ATG12. Cells were starved for 8 h or treated with CQ (50 µM) for 3 h. Scale bar: 10 µm. Image provided by M. Llanos Valero, M.A de la Cruz and R. Sanchez-Prieto

Figure 24.

Automated WIPI1 puncta image acquisition and analysis monitors the induction and inhibition of autophagy. Stable U2OS clones expressing GFP-WIPI1 were selected using 0.6 μg/ml G418 and then cultured in 96-well plates. Cells were treated for 3 h with nutrient-rich medium (Control), nutrient-free medium (EBSS), or with 233 nM wortmannin. Cells were fixed in 3.7% paraformaldehyde and stained with DAPI (5 μg/ml in PBS). An automated imaging and analysis platform was used to determine the number of both GFP-WIPI1 puncta-positive cells and the number of GFP-WIPI1 puncta per individual cell [664]. Cells without GFP-WIPI1 puncta are highlighted in red (cell detection) and purple (nuclei detection), whereas GFP-WIPI1 puncta-positive cells are highlighted in yellow (GFP-WIPI1 puncta detection), green (cell detection) and blue (nuclei detection). Bars: 20 µm. Images provided by S. Pfisterer and T. Proikas-Cezanne

Figure 25.

Clonal analysis of autophagy in the Drosophila larval midgut. Inhibition of autophagy in somatic clone cells marked by GFP (green, outlined) have decreased levels of mCherry-Atg8a puncta (red) compared to the control wild-type cells (non-GFP) with nuclei in blue (merged image, right panel). Bar: 20 µm. Image provided by D. Denton and S. Kumar

Figure 26.

S. cerevisiae cells were cultured to mid-log phase and shifted to SD-N for the indicated times. Samples were taken before (+) and at the indicated times after (–) nitrogen starvation. Immunoblotting was done with anti-phospho-Slt2 and anti-phospho-Hog1 antibody. This figure was modified from data previously published in ref. [747], and is reproduced by permission of the American Society for Cell Biology, copyright 2011

Figure 27.

Human fibroblasts showing colocalization of mitochondria with lysosomes. The degree of colocalization of mitochondria with lysosomes in human fibroblasts was measured via live cell imaging microscopy at 37°C and 5% CO2 atmosphere using the ApoTome® technique. LysoTracker® Red DND-99 staining was applied to mark lysosomal structures (red), and MitoTracker® Green FM to visualize mitochondria (green). Hoechst 33342 dye was used to stain nuclei (blue). A positive colocalization is indicated by yellow signals (Merge) due to the overlap of LysoTracker® Red and MitoTracker® Green staining (white arrows). Scale bar: 10 μm. Statistical evaluation is performed by calculating the Pearson’s coefficient for colocalizing pixels. Image provided by L. Burbulla and R. Krüger

Figure 28.

Detection of mitophagy in primary cortical neurons using mitochondria-targeted Keima. Neurons transfected with mito-Keima were visualized using 458-nm (green, mitochondria at neutral pH) and 561-nm (red, mitochondria in acidic pH) laser lines and 575-nm band pass filter. Compared with the control (A) wild-type PINK1 overexpression (B) increases the number of the mitochondria exposed to acidic conditions. Scale bar: 2 µm. (C) Quantification of red puncta suggests increased mitophagy in wild-type PINK1 but not in the kinase dead (kd) PINK1^K219M-overexpressing neurons. Image provided by V. Choubey and A. Kaasik

Figure 29.

PINK1-dependent phosphorylation of ubiquitin (p-S65-Ub) upon mitophagic stress. (A) Human dermal fibroblasts from healthy controls or PD patients carrying a PINK1 loss-of-function mutation (Q456X) were treated with valinomycin for the indicated times, and lysates were analyzed by western blot. The p-S65-Ub signal is almost undetectable under nonstress conditions in controls, but is strongly induced in a PINK1 kinase-dependent manner during its stabilization on the outer mitochondrial membrane. MFN2 serves as a control substrate and VCL (vinculin) as a loading control. (B) HeLa cells stably expressing GFP-PRKN (wild type) were treated with CCCP for the indicated times, fixed and stained with p-S65-Ub (red) and GFP-PRKN (green) as well as mitochondrial (TOMM20, cyan) and nuclear (Hoechst, blue) markers. The p-S65-Ub staining is almost undetectable in nonstressed cells, but rapidly accumulates on damaged mitochondria where it functions to activate PRKN. On mitochondria, PINK1 and PRKN together amplify the p-S65-Ub signal. Scale bar: 10 µm. Image provided by F.C. Fiesel and W. Springer

Figure 30.

Confocal microscopy deconvolved (AutoQuant X3) images and colocalization image analysis (ImageJ 1.47; Imaris 7.6) through a local approach showing perinuclear mitochondrial biogenesis in hippocampal neuronal cultures. The upper channels show TOMM20 (green channel), BrdU (for visualization of newly synthesized mitochondrial DNA, red channel), and merged fluorescence channels. Overlay, corresponds to the spatial pattern of software thresholded colocalized structures (white spots) layered on the merged fluorescence channels. Surface Plot, or luminance intensity height, is proportional to the colocalization strength of the colocalized structures (white spots). Plot Profile, corresponds to the spatial intensity profiles of the fluorescence channels of the white line positioned in the Merge image. Yellow arrows indicate a qualitative evaluation of the spatial association trends for the fluorescence intensities. Arrows pointing up indicate an increase in the colocalization, whereas arrows pointing down show a decrease. Scale bar: 2 μm. This figure was modified from previously published data [4085] and is provided by F. Florenzano

Figure 31.

Pathways that follow DNA damage may result in PCD or cell survival through autophagy, or checkpoint deficiency. (A) DNA damage inhibits TOR signaling, which promotes the formation of an ATG protein complex, thereby bringing about autophagy. Autophagy may contribute to cell survival as well as to cell death by PCD. (B) DNA damage may cause mutations in genes encoding checkpoint RAD proteins and/or proteins involved in DNA replication initiation, as well as the simultaneous deletion of genes encoding checkpoint kinases. Such protein defects lead to deficient checkpoints, thereby causing cells to enter M-phase prematurely with damaged DNA or incompletely replicated DNA, resulting in ROS generation, mitotic catastrophe, and subsequently PCD. This figure was previously published in ref. [1247]

Figure 32.

LysoTracker™ Red stains lysosomes and can be used to monitor autophagy in Drosophila. Live fat body tissues from Drosophila were stained with LysoTracker™ Red (red) and Hoechst 33342 (blue) to stain the nucleus. Tissues were isolated from fed (left) or 3-h starved (right) animals. Bar: 25 µm. This figure was modified from data presented in ref. [377], Developmental Cell, 7, Scott RC, Schuldiner O, Neufeld TP, Role and regulation of starvation-induced autophagy in the Drosophila fat body, pp. 167-78, copyright 2004, with permission from Elsevier

Figure 33.

GFP::LGG-1 and GFP::LGG-2 are autophagy markers in C. elegans. (A-F) Animals were generated that carry an integrated transgene expressing a GFP-tagged version of lgg-1, the C. elegans ortholog of mammalian MAP1LC3. Representative green fluorescence images in the pharyngeal muscles of (A) control RNAi animals without starvation, (B) control RNAi animals after 9 d of starvation, (C) atg-7 RNAi animals after 9 d of starvation, (D) starvation-hypersensitive gpb-2 mutants without leucine after 3 d of starvation, and (E) gpb-2 mutants with leucine after 3 d of starvation. The arrows show representative GFP::LGG-1-positive punctate areas that label pre-autophagosomal and autophagosomal structures. (F) The relative levels of PE-conjugated and unconjugated GFP::LGG-1 were determined by western blotting. These figures were modified from data previously published in ref. [2115], Kang, C., Y.J. You, and L. Avery. 2007. Dual roles of autophagy in the survival of C. elegans during starvation. Genes & Development. 21:2161-2171, Copyright © 2007, Genes & Development by Cold Spring Harbor Laboratory Press, and ref. [4086], Kang, C., and L. Avery. 2009. Systemic regulation of starvation response in C. elegans. Genes & development. 23:12-17, Copyright © 2011, Genes & Development by Cold Spring Harbor Laboratory Press, www.genesdev.org. (G-H) GFP:LGG-2 serves as a marker for autophagosomes in early C. elegans embryos. (G) GFP::LGG-2 expressed in the germline from an integrated transgene reveals the formation of autophagosomes (green) around sperm-inherited membranous organelles (red). DNA of the two pronuclei is stained (blue). (H) Later during development, GFP::LGG-2-positive structures are present in all cells of the embryo. Scale bar: 10 µm. Images provided by V. Galy

Figure 34.

Transmission electron micrograph of erythroblasts obtained from the blood of regular donors after ten days of culture in the presence of KITLG/SCF, IL3, EPO and dexamethasone. Original magnification 3000X. This figure shows two erythroblasts containing autophagic vacuoles. One erythroblast (red arrow) has the morphology of a live cell with several autophagic vacuoles that have engulfed cytoplasmic organelles. The other erythroblast (black arrow) has the electron-dense cytoplasm characteristic of a dead cell and is in the process of shedding its autolysosomes from the cytoplasm to the extracellular space. Image provided by A.R. Migliaccio and M. Zingariello

Figure 35.

A large dystrophic neurite from a brain biopsy of a patient with Gerstmann-Sträussler-Scheinker disease not unlike those reported for AD [79]. This structure is filled with innumerable autophagic vacuoles, some of which are covered by a double membrane. Electron dense lysosomal-like structures are also visible. The red arrow points to a double-membrane autophagic vacuole. Scale bar: 200 nm. Image provided by P. Liberski

Figure 36.

A high-power electron micrograph from a brain biopsy showing autophagic vacuoles in a case of ganglioglioma. Scale bar: 200 nm. Image provided by P. Liberski

Figure 37.

FIB-SEM images showing ultrastructural details of aging iPS cells. Arrows indicate autophagosomes containing mitochondria or other partially digested cytoplasmic material. E, exosomes; g, Golgi apparatus; m, mitochondrion; N, nucleus; RER, rough endoplasmic reticulum. Scale bars: 1 µm. Image provided by F. Colasuonno, modified from ref. [150]

Figure 38.

Autophagy in the digestive gland of Ruditapes decussatus (Mollusca, Bivalvia) subjected to a strict starvation of 2 months. Image provided by S. Baghdiguian

Figure 39.

Detection of autophagy in Echinococcus granulosus larval stage. (A) Scanning electron micrographs of a sectioned larva (or protoscolex) (i) showing big oval-shaped cells named calcareous corpuscles (red arrowheads) developed by cytoplasmic autophagy. Ultrastructural details of different developmental stages of these parenchymatic cells showing a central vacuole (ii-iii) at the initial development phase and concentric membranes that marginalize a thin layer of cytoplasm in mature corpuscles at the end of the autophagic process (iv-v). Energy-dispersive X-ray elemental microanalysis of the calcareous corpuscles in a sectioned protoscolex demonstrates the colocalization of accumulated ions into corpuscles: calcium (vi), phosphorus (vii). Scale bar: 10 µm. (B) Optical transmission (i) and confocal (ii-iv) microscopy images of a protoscolex treated with metformin (10 mM) for 48 h (i-iii) and an untreated microcyst (or metacestode) (iv) incubated with an anti-LC3 antibody and revealed with an antibody conjugated with Alexa Fluor 488 (green fluorescence) and counterstained with propidium iodide (red fluorescence) to observe cell nuclei under optimal contrast conditions. Fluorescent punctate images are often detected in the tegument of rapamycin-treated protoscoleces (ii-iii) and microcysts originated by vesicular de-differentiation from protoscoleces (iv) with high Atg8 polypeptide levels within the free cytoplasmic matrix of these cells, demonstrating pharmacological autophagy induction in corpuscles (ii-iii) and basal autophagy in small cysts in development even under nutrient-rich conditions (iv). Scale bar: 100 µm. Inset images correspond to TEM. bo, body; gl, germinal layer; su, sucker; tg, tegument. Images provided by A. C. Cumino and J. A. Loos. Only images in panel B were previously published in ref. [327]

Figure 40.

Detection of autophagy in tobacco BY-2 cells. (A) Induction of autophagosomes in tobacco BY-2 cells expressing YFP-NtAtg8 (shown in green for ease of visualization) under conditions of nitrogen limitation (Induced). Arrowheads indicate autophagosomes that can be seen as a bright green dot. No such structure was found in cells grown in normal culture medium (Control). Bar: 10 µm. N, nucleus; V, vacuole. (B) Ultrastructure of an autophagosome in a tobacco BY-2 cell cultured for 24 h without a nitrogen source. Bar: 200 µm. AP, autophagosome; CW, cell wall; ER, endoplasmic reticulum; P, plastid. Image provided by K. Toyooka

Figure 1.

Schematic model demonstrating the induction of autophagosome formation when turnover is blocked versus normal autophagic flux, and illustrating the morphological intermediates of autophagy. (A) The initiation of autophagy includes the formation and expansion of the phagophore, the initial sequestering compartment, which expands into an autophagosome. Completion of the autophagosome requires an intraphagophore membrane scission step and is followed by fusion of the outer autophagosomal membrane with lysosomes and degradation of the contents, allowing complete flux, or flow, through the entire pathway. This is a different outcome than the situation shown in (B) where induction results in the initiation of autophagy, but a defect in autophagosome turnover due, for example, to a block in fusion with lysosomes or disruption of lysosomal functions will result in an increased number of autophagosomes. In this scenario, autophagy has been induced, but there is no or limited autophagic flux. (C) An autophagosome can fuse with an endosome to generate an amphisome, prior to fusion with the lysosome. (D) Schematic drawing showing the formation of an autophagic body in fungi. The large size of the fungal vacuole relative to autophagosomes allows the release of the single-membrane autophagic body within the vacuole lumen. In cells that lack vacuolar hydrolase activity, or in the presence of inhibitors that block hydrolase activity, intact autophagic bodies accumulate within the vacuole lumen and can be detected by light microscopy. The lysosome of most more complex eukaryotes is too small to accommodate an autophagic body

Figure 2.

An autophagic body in a large lysosome of a mammalian epithelial cell in a mouse seminal vesicle in vitro. The arrow shows the single limiting membrane covering the sequestered rough ER. Image provided by A.L. Kovács

Figure 3.

TEM images of autophagic vacuoles in isolated mouse hepatocytes. (A) One autophagosome or early autophagic vacuole (AVi) and one degradative autophagic vacuole (AVd) are shown. The AVi can be identified by its contents (morphologically intact cytoplasm, including ribosomes, and rough ER), and the limiting membrane that is partially visible as two bilayers separated by a narrow electron-lucent cleft, i. e., as a double membrane (arrow). The AVd can be identified by its contents, partially degraded, electron-dense rough ER. The vesicle next to the AVd is an endosomal/lysosomal structure containing 5-nm gold particles that were added to the culture medium to trace the endocytic pathway. (B) One AVi, containing rough ER and a mitochondrion, and one AVd, containing partially degraded rough ER, are shown. Note that the limiting membrane of the AVi is not clearly visible, possibly because it is tangentially sectioned. However, the electron-lucent cleft between the two limiting membranes is visible and helps in the identification of the AVi. The AVd contains a region filled by small internal vesicles (asterisk), indicating that the AVd has fused with a multivesicular endosome. mi, mitochondrion. Image provided by E.-L. Eskelinen

Figure 4.

Autophagosomes with recognizable cargo are rare in cells. (A) To assess relative rates of autophagosome formation, the lysosomal inhibitor bafilomycin A₁ (10 nM) was applied for 2 h prior to fixation with 2% glutaraldehyde in order to trap newly formed autophagosomes (note that whereas short-term treatment with bafilomycin A₁ in most cases primarily blocks autolysosomal degradation, it can also inhibit autophagosome-lysosome fusion). Two different PINK1 shRNA lines (A14 and D14) exhibit increased AV formation over 2 h compared to the control shRNA line. *, p < 0.05 vs. Control. (B) Autophagosomes in bafilomycin A₁-treated control cells contain a variety of cytoplasmic structures (left, arrow), whereas mitochondria comprise a prominent component of autophagosomes in bafilomycin A₁-treated (PINK1 shRNA) cells (right, arrow). Scale bar: 500 nm. These data indicate induction of selective mitophagy in PINK1-deficient cells. This figure was modified from Figure 2 published in Chu CT. A pivotal role for PINK1 and autophagy in mitochondrial quality control: implications for Parkinson disease. Human Molecular Genetics 2010; 19:R28-R37

Figure 5.

Cryoelectron microscopy can be used as a three-dimensional approach to monitor the autophagic process. Computed sections of an electron tomogram of the autophagic vacuole-rich cytoplasm in a hemophagocyte of a semi-thin section after high-pressure freezing preparation. The dashed area is membrane-free (A) but tomography reveals newly formed or degrading membranes with a parallel stretch (B). Image published previously [4082] and provided by M. Schneider and P. Walter

Figure 6.

Different autophagic vacuoles observed after freeze fracturing in cultured osteosarcoma cells after treatment with the autophagy inducer voacamine [143]. (A) Early autophagosome delimited by a double membrane. (B) Inner monolayer of an autophagosome membrane deprived of protein particles. (C) Autolysosome delimited by a single membrane rich in protein particles. In the cross-fractured portion (on the right) the profile of the single membrane and the inner digested material are easily visible. Images provided by S. Meschini, M. Condello and A. Giuseppe

Figure 7.

LC3-I conversion and LC3-II turnover. (A) Expression levels of LC3-I and LC3-II during starvation. Atg5^+/+ (wild-type) and atg5^−/− MEFs were cultured in DMEM without amino acids and serum for the indicated times, and then subjected to immunoblot analysis using anti-LC3 antibody and anti-tubulin antibody. E-64d (10 µg/ml) and pepstatin A (10 µg/ml) were added to the medium where indicated. Positions of LC3-I and LC3-II are marked. The inclusion of lysosomal protease inhibitors reveals that the apparent decrease in LC3-II is due to lysosomal degradation as easily seen by comparing samples with and without inhibitors at the same time points (the overall decrease seen in the presence of inhibitors may reflect decreasing effectiveness of the inhibitors over time). Monitoring autophagy by following steady-state amounts of LC3-II without including inhibitors in the analysis can result in an incorrect interpretation that autophagy is not taking place (due to the apparent absence of LC3-II). Conversely, if there are high levels of LC3-II but there is no change in the presence of inhibitors this may indicate that induction has occurred but that the final steps of autophagy are blocked, resulting in stabilization of this protein. This figure was modified from data previously published in ref. [30], and is reproduced by permission of Landes Bioscience, copyright 2007. (B) Lysates of four human adipose tissue biopsies were resolved on two 12% polyacrylamide gels, as described previously [292]. Proteins were transferred in parallel to either a PVDF or a nitrocellulose membrane, and blotted with anti-LC3 antibody, and then identified by reacting the membranes with an HRP-conjugated anti-rabbit IgG antibody, followed by ECL. The LC3-II:LC3-I ratio was calculated based on densitometry analysis of both bands. *, P< 0.05. (C) HEK 293 and HeLa cells were cultured in nutrient-rich medium (DMEM containing 10% fetal calf serum) or incubated for 4 h in starvation conditions (Krebs-Ringer medium) in the absence (-) or presence (+) of E-64d and pepstatin at 10 µg/ml each (Inhibitors). Cells were then lysed and the proteins resolved by SDS-PAGE. Endogenous LC3 was detected by immunoblotting. Positions of LC3-I and LC3-II are indicated. In the absence of lysosomal protease inhibitors, starvation results in a modest increase (HEK 293 cells) or even a decrease (HeLa cells) in the amount of LC3-II. The use of inhibitors reveals that this apparent decrease is due to lysosome-dependent degradation. This figure was modified from data previously published in ref. [240], and is reproduced by permission of Landes Bioscience, copyright 2005. (D) Sequence and schematic representation of the different forms of LC3B. The sequence for the nascent (proLC3) from mouse is shown. The glycine at position 120 indicates the cleavage site for ATG4. After this cleavage, the truncated LC3 is referred to as LC3-I, which is still a soluble form of the protein. Conjugation to PE generates the membrane-associated LC3-II form (equivalent to Atg8–PE)

Figure 8.

Different LC3B-I:LC3B-II ratios indicating turnover were assessed using a mono- as well as polyclonal anti-LC3B antibody. Monocytes were isolated from human whole blood and differentiated into monocyte-derived macrophages (MDMs) by incubation in human CSF1/M-CSF for 1 week. To induce autophagy, cells were starved by reducing the FCS concentration to 1% for one day. Monocytes, and resting and starved MDMs were lysed with Laemmli buffer; the proteins were separated by SDS-PAGE and analyzed by western blot. Membranes were labeled using a monoclonal antibody to the N terminus of LC3B (Novus, clone 1251D, NBP2-59800) or polyclonal antibodies (Sigma, L7543). Relative intensity of LC3B-I and LC3B-II was quantified with Image Lab™ to calculate LC3B-II:LC3B-I ratios

Figure 9.

Detection of nonlipidated (LC3-I) and lipidated (LC3-II) forms of the LC3 protein using (A) traditional SDS-PAGE and western blotting or (B) the WES System (WES - Automated Western Blots with Simple Western; ProteinSimple, San Jose, CA, USA). HEK 293 cells were cultured in DMEM medium, containing 10% fetal bovine serum and a penicillin-streptomycin mixture, at 37°C in a humidified atmosphere with 5% CO₂. Cell cultures were treated with medium devoid of fetal bovine serum (-FBS) or with chloroquine (CQ; final concentration 10 µM) for 2 h. The forms of the LC3 protein were detected using anti-LC3 antibodies (MBL International, PM036). Materials from the same samples were used in experiments presented in panels A and B. The LC3-I and LC3-II forms can be effectively separated using traditional SDS-PAGE and western blotting, whereas these two forms of LC3 cannot be distinguished by using the WES system. Results provided by K. Pierzynowska, L. Gaffke and G. Wegrzyn

Figure 10.

Measuring autophagic flux and pool size of pathway intermediates at the single-cell level: autophagosome, autolysosome and lysosome pool size and flux data, characterizing MEF cells with a basal flux of 25 autophagosomes/h/cell, which increases upon rapamycin treatment to 105 autophagosomes/h/cell. Scale bar: 20 µm. This figure was previously published in ref. [312]

Figure 11.

Effect of different inhibitors on LC3-II accumulation. SH-SY5Y human neuroblastoma cells were plated and allowed to adhere for a minimum of 24 h, then treated in fresh medium. Treatments were as follows: rapamycin (Rap), (A) 1 µM, 4 h or (B) 10 µM, 4 h; E-64d, final concentration 10 µg/ml from a 1 mg/ml stock in ethanol (EtOH); NH₄Cl (NH₄⁺), final concentration 10 mM from a 1 M stock in water; pepstatin A (Pst), final concentration 10 µg/ml from a 1 mg/ml stock in ethanol, or 68.6 µg/ml from a 6.86 mg/ml stock in DMSO; ethanol or DMSO, final concentration 1%. Pre-incubations in (B) were for 1 or 4 h as indicated. 10 mM NH₄Cl (or 30 µM CQ, not shown) were the most effective compounds for demonstrating the accumulation of LC3-II. E-64d was also effective in preventing the degradation of LC3-II, with or without a preincubation, but ammonium chloride (or CQ) may be more effective. Pepstatin A at 10 µg/ml with a 1-h pre-incubation was not effective at blocking degradation, whereas a 100 µM concentration with 4-h pre-incubation had a partial effect. Thus, alkalinizing compounds are more effective in blocking LC3-II degradation, and pepstatin A must be used at saturating conditions to have any noticeable effect. Images provided by C. Isidoro. Note that the band running just below LC3-I at approximately 17.5 kDa may be a processing intermediate of LC3-I; it is detectable in freshly prepared homogenates, but is less visible after the sample is subjected to a freeze-thaw cycle

Figure 12.

GFP-LC3 processing can be used to monitor delivery of autophagosomal membranes. (A) atg5^−/− MEFs engineered to express Atg5 under the control of the Tet-off promoter were grown in the presence of doxycycline (Dox; 10 ng/ml) for one week to suppress autophagy. Cells were then cultured in the absence of drug for the indicated times, with or without a final 2-h starvation. Protein lysates were analyzed by western blot using anti-LC3 and anti-GFP antibodies. The positions of untagged and GFP-tagged LC3-I and LC3-II, and free GFP are indicated. This figure was modified from data previously published in ref. [346], FEBS Letters, 580, Hosokawa N, Hara Y, Mizushima N, Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size, pp. 2623-2629, copyright 2006, with permission from Elsevier. (B) Differential role of unsaturating and saturating concentrations of lysosomal inhibitors on GFP-LC3 cleavage. HeLa cells stably transfected with GFP-LC3 were treated with various concentrations of CQ for 6 h. Total lysates were prepared and subjected to immunoblot analysis. (C) CQ-induced free GFP fragments require classical autophagy machinery. Wild-type and atg5^−/− MEFs were first infected with adenovirus GFP-LC3 (100 viral particles per cell) for 24 h. The cells were then either cultured in regular culture medium with or without CQ (10 µM), or subjected to starvation in EBSS buffer in the absence or presence of CQ for 6 h. Total lysates were prepared and subjected to immunoblot analysis. Panel B and C are modified from the data previously published in ref. [351]

Figure 13.

Movement of activated pDendra2-hp62 (SQSTM1; orange) from the nucleus (middle) to an aggregate in ARPE-19 cells, revealed by confocal microscopy. Cells were exposed to 5 µM MG132 for 24 h to induce the formation of perinuclear aggregates [4083]. The cells were then exposed to a UV pulse (the UV-induced area is shown by red lines that are inside of the nucleus) that converts Dendra2 from green to red, and the time shown after the pulse is indicated. SQSTM1 is present in a small nuclear aggregate, and is shuttled from the nucleus to a perinuclear large protein aggregate (detected as red). Scale bar: 5 µm. Image provided by K. Kaarniranta

Figure 14.

The HaloTag-LC3 assay distinguishes phagophores, immature autophagosomes, and mature autophagosomes and autolysosomes. (A) Schematic diagram of the HaloTag-LC3 (HT-LC3) assay. Cells expressing HT-LC3 are treated with a cholesterol-dependent plasma membrane permeabilizer to release cytosolic proteins including HT-LC3-I and sequentially labeled with a saturated dose of membrane-impermeable HaloTag ligands (MILs) conjugated with Alexa Fluor 488 (or 660) followed by membrane-permeable HaloTag ligands (MPLs) conjugated with tetramethylrhodamine to detect phagophores (MIL⁺ MPL⁻), immature autophagosomes (iAP; MIL⁺ MPL⁺), and mature autophagosomes and autolysosomes (AP and AL; MIL⁻ MPL⁺). (B) U-2 OS cells were stably transduced with HT-LC3-encoding lentiviruses, incubated in starvation medium or control complete medium in the presence or absence of 100 nM bafilomycin A₁ (BafA1) for 4 h, and subjected to the HT-LC3 assay followed by confocal microscopy. Magnified images of the boxed (i) and arrow-indicated (ii-v) areas are shown in the right panels. Scale bars: 10 μm (1 μm in the magnified images)

Figure 15.

Changes in the detection and localization of GFP-LC3 upon the induction of autophagy. U87 cells stably expressing GFP-LC3 were treated with PBS (Control), rapamycin (200 nM), or rapamycin in combination with 3-MA (2 mM) for 24 h. Representative fluorescence images of cells counterstained with DAPI (nuclei) are shown. Scale bar: 10 µm. This figure was modified from Figure 6 published in ref. [364], Badr et al. Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway. Neuro-Oncology, 13(11):1213-24, 2011, by permission of Oxford University Press

Figure 16.

The GFP and mRFP signals of tandem fluorescent LC3 (tfLC3, mRFP-GFP-LC3) show different localization patterns. HeLa cells were cotransfected with plasmids expressing either tfLC3 or LAMP1-CFP. Twenty-four h after the transfection, the cells were starved in Hanks balanced salt solution for 2 h, fixed and analyzed by microscopy. The lower panels are a higher magnification of the upper panels. Bar: 10 µm in the upper panels and 2 µm in the lower panels. Arrows in the lower panels point to (or mark the location of) typical examples of colocalized signals of mRFP and LAMP1. Arrowheads point to (or mark the location of) typical examples of colocalized particles of GFP and mRFP signals. This figure was previously published in ref. [344], and is reproduced by permission of Landes Bioscience, copyright 2007

Figure 17.

GFP fluorescence in the autolysosome can be recovered upon neutralization of the pH. (A) GFP-LC3 emits green fluorescence in the autolysosomes of post-mortem processed heart sections. Cryosections of 3.8% paraformaldehyde fixed ventricular myocardium from 3-week-old GFP-LC3 transgenic mice at the baseline (Control) or starved for 24 h (Starved) were processed for immunostaining using a standard protocol (buffered at pH 7.4). Most of the GFP-LC3 puncta are positive for LAMP1, suggesting that the autolysosomes had recovered GFP fluorescence. (B) Colocalization between GFP-LC3 direct fluorescence (green) and indirect immunostaining for GFP (red). Sections processed as in (A) were immunostained for GFP using a red fluorescence-tagged secondary antibody, and the colocalization with GFP fluorescence was examined by confocal microscopy. Almost all of the red puncta emit green fluorescence. Scale bar: 10 µm. Image provided by Xuejun Wang

Figure 18.

Saponin extraction allows quantification of LC3-II fluorescence by FACS. (A) Schematic diagram of the effects of the saponin wash. Due to the reorganization of the EGFP-LC3 reporter protein, induction of autophagosome formation does not change the total levels of fluorescence in EGFP-LC3-transfected cells. However, extraction of EGFP-LC3-I with saponin results in a higher level of fluorescence in cells with proportionally higher levels of EGFP-LC3-II-containing autophagosomes. This figure was previously published in ref. [433]. (B) Saponin extraction can also be used to measure flux of endogenous LC3 protein. Human osteosarcoma cells were starved of amino acids and serum by incubation in EBSS, for the indicated times in the presence or absence of a 1-h CQ (50 μM) treatment. Cells were then washed with PBS containing 0.05% saponin and processed for FACS analysis for endogenous LC3. Image provided by K.E. Eng and G.M. McInerney