Induction of macroautophagy by exogenously introduced calcium

Abstract

Macroautophagy can be activated by a broad range of agents and cellular manipulations. In performing cellular transfection using the calcium phosphate method, we noticed that the calcium phosphate precipitates (CPP) could induce LC3 punctation. Because of the wide use of this transfection method in mammalian cells and the potential significance of calcium in autophagy induction, we investigated whether CPP could specifically induce macroautophagy. We found that CPP-induced LC3 punctation was dependent on calcium and could be neutralized by extracelluar or intracellular calcium chelator. The punctation was not due to non-specific aggregation of LC3 since it depended on the amino acid residue Glycine120, which is specifically required for LC3 to conjugate to phosphatidylethanolamine (PE). Consistently, there was also a significant increase of the PE-conjugated form of LC3. Electron microscopy confirmed the accumulation of typical autophagosomes following CPP treatment. Flux analysis indicated that CPP induced but did not inhibit autophagic degradation. Finally CPP-induced autophagy depended on the classical macroautophagy machinery including Beclin 1, the class III phosphoinositide-3 kinase and Atg5. Our studies thus indicate that exogenously introduced calcium in the form of CPP could specifically induce macroautophagy, which may have the practical significance in the use of this agent for introducing genes into cells, and for studying the mechanism of autophagy as a model system.

Introduction

Autophagy is an intracellular degradation system, which is responsible for the catabolism of proteins and subcellular organelles in the lysosome ^{1, 2}. Three types of autophagy have been defined, namely macroautophagy, microautophagy and chaperone-mediated autophagy. They differ in the way by which the substrates are delivered to the lysosome and the corresponding molecular machinery. In the macroautophagy (hereafter referred to as autophagy), the substrates are delivered by the autophagosomes.

Thirty-one genes have been defined that participate in macroautophagy or autophagy-related process in the yeast, many of which have mammalian homologs ^{2, 3}. The core machinery seems to be built around two ubiquitin-like conjugation systems ⁴. In one system, the ubiquitin-like protein, Atg12, is first activated by Atg7, an ubiquitin-activating enzyme (E1)-like protein, and then transferred by Atg10, an ubiquitin carrier protein (E2)-like protein, to Atg5 through a covalent bond. The Atg5-Atg12 complex interacts with Atg16 to form a multimer complex, which is translocated to membranes of early autophagosomes. In another system, a ubiquitin-like protein, Atg8, or one of its mammalian orthologs, the microtubule-associated protein 1 light chain 3 (LC3), is first cleaved by Atg4 to expose the conserved Gly¹²⁰ at its C-terminus. Atg8/LC3 is then conjugated to phosphatidylethanolamine (PE), via Atg7 and Atg3, another ubiquitin carrier protein (E2)-like protein ^{4, 5}. The un-conjugated form of Atg8/LC3 (called LC3-I) is in the cytosol while the conjugated form (called LC3-II) targets to the autophagosomal membrane ⁵ following the Atg5-Atg12-Atg16 complex. This association of Atg8/LC3-PE at the autophagosomal membrane is considered important for the membrane extension of the autophagosome and the eventual enclosure of the membrane to form the vesicle. Genetic deletion of Atg 5 or Atg7 in mice has revealed the non-redundant function of these molecules in mammalian autophagy ^{6, 7}.

Several other key autophagy genes are important to the initiation and regulation of autophagy. Atg6 and its mammalian homolog, Beclin 1, is particularly important. It forms a complex with VPS34 and several other proteins to function as a key initiation mechanism. VPS34 is a class III phosphoinositide (PI)-3-kinase required for autophagy, which can be inhibited by 3-methyladenine (3-MA) ⁸. While the actual mechanism of this complex is not yet known, deletion of Beclin 1 in mammalian cells leads to the blockage of autophagy, likely at an upstream point ⁹.

Autophagy is activated and regulated by many physiological and pathological conditions and in turn affects these processes ¹⁰. Autophagy is important for the regulation of energy and nutrient metabolism ^{11, 12}. In addition, degradation of mitochondria, peroxisomes, endoplasmic reticulum (ER), or ribosomes by autophagy is most likely associated with cellular homeostasis as well as the changing metabolic needs ^{1, 2, 10}. The ability of autophagy to degrade misfolded proteins is an important beneficial function in the pathogenesis of conformational diseases ^{13, 14}. Autophagy could play important roles in cancer development and in cancer therapy ^{15, 16}. Finally autophagy has been shown to be critical in innate immunity to control intracellular microbial infection ¹⁰.

An important task in autophagy study is to define the various signal transduction pathways that link the stimuli to the core autophagic machinery. In addition, defining novel chemicals that can either stimulate or suppress autophagy could have significances in both clinical applications and in the understanding of autophagy mechanisms. Toward that end, we studied the autophagy-inducing activity of a unique chemical substance, calcium phosphate precipitates (CPP), which is used in a common transfection procedure in mammalian cells. The characterization of CPP-induced autophagy would also provide a model to study the mechanism of autophagy, particularly that induced by calcium.

Results

Calcium phosphate precipitates induced specific LC3 punctation in a dose-dependent manner

During the work in introducing DNA into the cell by the calcium phosphate method, we noticed the induction of LC3 punctation during the process. Because of the potential significance of this phenomenon in the understanding of how calcium may induce autophagy, we conducted a detailed study to characterize it. The calcium phosphate transfection procedure was one of the most common ways to introduce DNA into the mammalian cells. It relies on the calcium phosphate precipitates (CPP) formed by the interaction of calcium chloride (CaCl₂) and sodium phosphate dibasic (Na₂HPO₄) with DNA binding to the calcium ions. However, DNA is not required for precipitates to form. In a standard transfection procedure, the precipitates are usually formed with a final concentration of 128 mM of CaCl₂ and 0.75 mM of Na₂HPO₄. We found that when this solution was added to culture in a 1:10 dilution, i.e., 100 μl per ml of culture, about 10% of HEK293 cells that were stably expressing GFP-LC3 would transiently demonstrated large LC3 punctation, while little or no LC3 puncta could be detected at the basal non-stimulated status. Subsequent testing ruled out the role of DNA in this process and suggested that LC3 punctation was due solely to CPP.

To better characterize the effect of CPP, we mixed an equal volume of calcium chloride solution (256 mM) with a phosphate buffer containing 3 mM of Na₂HPO₄. While the final concentration of CaCl₂ (128 mM) was the same as used in the standard transfection, the final concentration of Na₂HPO₄ (1.5 mM) was raised based on pilot study (data not shown) to increase the potential amount of precipitates and to reduce the total volume of the solution to be added to the culture. The CPP-containing solution was then added to cell at different amounts. A rapid induction of LC3 puncta could be observed when about 50 μl of CPP solution per milliliter of culture was added (Fig. 1A-C). The kinetics and the extent of the induction were dose-dependent with a higher dose of CPP giving rise to a more rapid induction in more cells for a longer duration (Fig. 1D). In a typical experiment a dose of 100 μl of CPP solution per milliliter of culture was able to induce GFP-LC3 punctation in almost 100% of cells in 6−8 hours, which, however, subsided to the basal level in 24 hours. Cell viability did not seem to be noticeably affected during this period (data not shown). This induction of LC3 punctation was clearly mediated by calcium signaling, since the Na₂HPO₄ buffer (Buffer A) alone did not induce such changes (data not shown, also see below) and more importantly, treatment of cells with an extracelluar calcium chelator, EGTA, or an intracellular calcium chelator, BAPTA-AM, completely inhibited the response (Fig. 1E).

Figure 1. Calcium phosphate precipitates could induce a dose-dependent LC3 punctation.

(A). 293 cells expressing GFP or GFP-LC3 (wild type or G120A mutant) in complete medium or treated with CPP (100 μl/ml) for 6 hours. Numbers indicate the percentage of cells with LC3 punctation. (B). 293 cells expressing RFP or RFP-LC3 (wild type or G120A mutant) in complete medium or treated with CPP (100 μl/ml) for 6 hours. Numbers indicate the percentage of cells with large LC3 ring formation. (C). HCT116-(Bax −/−) cells expressing GFPLC3 were treated with control or CPP (100 μl/ml) for 6 hours. Numbers indicate the percentage of cells with LC3 punctation. (D). 293 cells expressing GFP-LC3 (wild type) were treated with different amounts of CPP (25 μl to 200 μl per 1 ml of culture medium) for different times as indicated. The percentage of cells showing GFP-LC3 punctation was determined. (E). 293-GFP-LC3 cells treated with CPP (100 μl/ml) for 3 hours with or without EGTA (3 mM). Alternatively, cells were pre-treated with BAPTA-AM (40 μM) for 30 minutes before CPP was added. The percentage of cells showing GFP-LC3 punctation was determined. (F). 293 cells were treated with or without CPP (100 μl/ml) for 6 hours and then fixed. Cells were immunostained with an anti-LC3 antibody followed by Cy-3-conjugated secondary antibody. All data shown are mean±SD from one representative experiment of at least three performed.

The formation of LC3 puncta was specific to the autophagy process and required an autophagy-competent form of LC3, since the G120A mutant of LC3 could not form puncta following CPP treatment (Fig. 1A, B). Gly120 of human LC3 is evolutionarily conserved and is required for LC3 cleavage by Atg4, which in turn allows LC3 to be conjugated to PE and targeted to autophagosomes ⁴. We had also confirmed that this punctation of LC3 was not related to the specific type of fluorescence molecule it was fuse to. Thus either GFP-LC3 (Fig. 1A) or RFP-LC3 (Fig. 1B) responded to CPP treatment in Gly120-specfical manner. Furthermore, endogenous LC3 was also found to form puncta following CPP treatment as revealed by immunostaining (Fig. 1F). Finally while initial studies were conducted in HEK293 cells, the CPP-induced LC3 punctation could be seen in other types of cells, including the HCT116 colon cancer cells (Fig. 1C) and murine embryonic fibroblasts (MEFs) (see below Figure 5).

Figure 5. CPP-induced macroautophagy depended on Atg5.

Wild type and Atg5-deficient MEFs were infected with Ad-GFP-LC3 for 16 hours and then treated with CPP (100 μl/ml) for 6 hours. The percentages (mean±SD) of cells exhibiting LC3 punctation were indicated for each treatment and cell line.

Calcium phosphate precipitates induced macroautophagy

To determine the significance of LC3 punctation and to further rule out any non-specificity, we performed electron microscopy on CPP-treated cells. We found that there was a significant increase in autophagosome formation following the treatment (Fig. 2). Typical autophagosomes with double membranes and cellular contents could be readily detected. Thus the accumulation of LC3 punctation was correlated with the increase in autophagosomes, stimulated by CPP.

Figure 2. Calcium phosphate precipitates induced autophagosome formation.

293 cells were untreated (A) or treated with CPP (200 μl/ml) for 6 hours (B). Cells were then processed and examined by electron microscopy. Boxed area in panel B was further enlarged. Black arrows indicate the autophagosomes with double membranes while the white arrowhead denotes an autolysosome. Bar = 2 micron, N, nucleus. (C). The number of autophagic vesicles (AV) per cell section (mean±SD) was determined from 20 cells of control and treated samples, respectively.

To determine whether this accumulation of autophagosomes was due to an increased induction of autophagy or to a blockage of basal level autophagosome degradation, we examined several parameters that are related to the autophagy flux ¹⁷. CPP stimulation of GFP-LC3 punctation was accompanied by the appearance of the PE-conjugated form of LC3 (LC3-II)(Fig. 3A). The lipidation could be also demonstrated in the endogenous LC3 following the treatment with CPP, but not with Buffer A (Fig. 3B). More importantly, immunoblot analysis of the GFPLC3-II form clearly showed a time-dependent reduction with the gradual appearance of the GFP moiety alone (Fig. 3A). Previous study had demonstrated that this indicated the degradation of the PE-conjugated LC3 moiety in the lysosome with the survival of the relatively hydrolysis-resistant GFP moiety ¹⁷. Indeed, inhibition of lysosome function with chloroquine led to the reduction of this degradation and of GFP accumulation.

Figure 3. Calcium phosphate precipitates promoted autophagic degradation.

(A). 293 cells expressing GFP or GFP-LC3 were treated as indicated with or without CPP (100 μl/ml) for the indicated time with or without chloroquine (CQ, 40 μM). Immunoblot was conducted with anti-GFP, anti-p62 and anti-β-actin antibodies. This is one representative experiment of at least three performed. (B). 293 cells were incubated complete medium (CM), or treated with Buffer A or CPP (100 μl/ml) for 6 hours. Cells were lysed and the lysates were subjected to immunoblot analysis with an anti-LC3 or anti-β-actin antibody. The positions of the I and II forms of the endogenous LC3 are indicated. (C). 293 cells stably expressing RFP-GFP-LC3 was treated with medium (Control) or CPP (100 μl/ml) for 6 hours and the signals in the green and red channels were obtained and merged. In control samples, small puncta in the RFP channel may indicate the basal level of autophagy in this line, which would not be detected in GFP channel. In CPP-treated samples, induced puncta were larger and could be ring-shaped. Many of the red puncta were again free of green colors. Arrows indicates typical dots that likely represented the later stage of degradation in the acidic compartment. (D). The number of LC3 positive puncta (small dots and large ring-shaped puncta) per cell was quantified on the green channel (Green Puncta) or the red channel (Red Puncta) in control and CPP-treated cells. Data shown are mean±SD from 30 cells of each group.

A LC3-binding molecule, p62/SQSTM1, is important for autophagic clearance of certain proteins and seems to be degraded through the autophagy pathway as it accumulates significantly in autophagy-deficient cells ¹⁷. We found that the degradation of p62 was enhanced by CPP, which could be blocked when lysosome function was suppressed (Fig. 3A), in parallel to the changes of GFP-LC3-II and GFP.

Finally, we introduced a newly developed tool for analyzing autophagy flux, the tandem RFP-GFP-LC3 construct ¹⁸. This construct fuses the LC3 molecule with both RFP and GFP in tandem. The GFP is more prone than RFP to the acidic environment of lysosomes. Thus the RFP signals tend to last longer than GFP signals. By comparing the GFP (in the green channel) versus RFP fluorescence signals (in the red channel), it is possible to determine whether accumulated LC3, therefore the autophagosomes so labeled, had entered into the lysosome compartment or not. A 293 stable cell line expressing RFP-GFP-LC3 was established. As reported previously, under the basal level there was few LC3 punctation based on GFP signals, but such punctation could be detected in the RFP channel, indicating the degradation of LC3 in the autolysosomes due to the basal autophagic activity (Fig. 3C-D). When the cells were stimulated with CPP, an increased LC3 punctation, including some large-sized ring shaped structure, could be clearly detected in the GFP channel (Fig. 3C-D), consistent with the finding with the single GFP-LC3 construct (Fig. 1A). However, additional puncta could be detected in the RFP channel, suggesting that some of the newly induced LC3 puncta, i.e., autophagosomes, had moved into the later stage of degradation (Fig. 3C-D).

Together these findings indicated that CPP induced a significant level of autophagy rather than blocking the basal level of autophagosome degradation.

Calcium phosphate precipitates induced macroautophagy through the classical autophagy pathway

The classical macroautophagy pathway involves the participation of Beclin 1/Atg6, which forms an autophagy specific complex with the class III PI-3 kinase, VPS34, its regulatory partner, p150/VPS15, and Atg14. The complex is critical to the function of the Atg12-Atg5 conjugation system, which is in turn important for the LC3-PE conjugation and for the autophagosome formation. A commonly used VPS34 inhibitor, 3-MA or a Beclin 1 specific siRNA could significantly suppress CPP-induced LC3 punctation, GFP-LC3-II formation and its degradation (Fig. 4). Consistently, deletion of Atg5 resulted in a complete blockage of CPP-induced LC3 punctation (Fig. 5). These pharmacological, molecular and genetic manipulations confirmed that CPP can activate the classical autophagy pathway to induce macroautophagy.

Figure 4. CPP-induced macroautophagy depended on Beclin-1 and Class III PI-3 kinase.

(A). 293-GFP-LC3 treated with medium or CPP (100 μl/ml) for 6 hours with or without 3-MA (20 mM). Alternatively cells were transfected with negative or Beclin 1-specific siRNA (si-Bec 1) and then treated with CPP (100 μl/ml) for 2 hours. Percentages (mean±SD) of cells exhibiting GFP-LC3 punctation were shown. Data were from one representative experiment of at least three performed. (B). Immunoblot analysis of Beclin-1 and β-actin expression in control siRNA-transfected cells (lane 1) or in Beclin-1 specific siRNA transfected cells (lane 2). (C). 293-GFP-LC3 cells were treated as indicated for 6 hours and analyzed by immunoblot assay with an anti-GFP or β-actin antibody.

Discussion

In this study we demonstrated with convincing evidence that CPP-induced macroautophagy is specific and relies on classical autophagy machinery. LC3 punctation has been widely used as an indicator for autophagy induction ⁵. However, several recent studies found that LC3 punctation could be formed independently of autophagy due to LC3 incorporation into protein aggregates or transient over-expression ¹⁹. Permeabilization by many types of detergents, such as saponin, CHAPS, Triton X-100 or digitonin could also induce non-specific LC3 punctation ²⁰. In these cases, the LC3 punctation was either independent of Atg5 or not accompanied with the formation of the PE-conjugated LC3-II form. However, in the case of CPP-induced LC3 punctation in stable cell lines, we showed by a genetic, molecular and pharmacological approach that Beclin 1, Class III PI-3 kinase and Atg5 were all required. In addition, the dependence on Gly120 and the concomitant formation of LC3-II could be clearly demonstrated. Moreover, electron microscopic studies confirmed the accumulation of autophagic vesicles and flux analysis indicated an increased autophagic degradation upon CPP treatment. Based on these criteria ¹⁷ it is certain that CPP can specifically induce macroautophagy.

Calcium phosphate transfection is one of the earliest developed ways to introduce DNA into mammalian cells and it is still in wide use. It is simple, inexpensive, nontoxic and sufficiently efficient for many types of cells. However, it now seems that calcium phosphate precipitates are able to induce macroautophagy. It needs to point out, however, that the dosage of Na₂HPO₄ used in a standard transfection recipe is about half of what had been typically used here (0.75 mM vs 1.5 mM after mixing with the calcium chloride solution). A higher concentration of Na₂HPO₄ likely increased the extent of precipitation, although the actual amount of CPP would be difficult to determine. Nevertheless, CPP-induced LC3 punctation was positively correlated with the volume of CPP solution added to the culture. Importantly, this induction of LC3 punctation was transient and lasted only for 24−48 hours in the dose range used in this study (Fig. 1D). The potential significance of these findings may be that care should be exercised when autophagy studies are conducted with exogenous genes being introduced into cells using calcium phosphate transfection. At the standard transfection dose, CPP induce autophagy only in a small percentage of cells. Thus the interference from CPP could be avoided if the amount of Na₂HPO₄ does not exceed the recommended one and if the subsequent studies could be conducted 24 hours later or when GFP-LC3 punctation subsides after transfection if this reporter is used.

Other types of transient transfection, such as lipotransfection, might also transiently induce LC3 punctation, perhaps due to the toxicity of the transfection reagent and/or cellular stress incurred during the transfection. While these conditions have yet to be further characterized, it is generally advised that stably cell lines are used to assess LC3 punctation if a LC3 reporter construct is used ¹⁷.

A potentially more interesting issue with CPP is how this substance may induce macroautophagy. It is not clear how CPP, or CPP-DNA complex, is taken into the cells. Whatever the way it is, it may not be very efficient, as many cells could not be successfully transfected using this method. However, it seems that the effective component is the calcium ion, and that it has to be taken into the cells in order to induce autophagy. We found that simply increasing calcium concentration in the medium had no autophagic effects since it could not freely enter into cells under normal condition. In addition, adding EGTA in the medium at the time when CPP was applied would block the induction, suggesting that the calcium chelator either blocked the entrance of CPP into cells or rendered the calcium ion ineffective inside the cell. The latter possibility was confirmed when a cell permeable calcium chelator, BAPTA-AM, was added before CPP application, which could then chelate the calcium inside the cells.

Calcium has long been noticed to play a role in autophagy, although whether it promotes or inhibits autophagy may depend on actual cellular context. An early study suggested that intracellularly stored calcium was required for autophagic activity in hepatocytes ²¹. More recently, it was found that in a number of cases where calcium was released from endoplasmic reticulum it participated in autophagy induction by activating calmodulin-dependent kinase kinase-beta (CaMKKβ) and AMP activated protein kinase (AMPK), which can inhibit mTOR ²². Calcium could also activate other mechanisms in activating autophagy, e.g. by activating calpain. Calpain has been shown to be instrumental in autophagy induced by multiple signals, such as starvation, rapamycin and ceramide, in fibroblasts ²³. However calcium and calpain could be found in other scenarios to be anti-autophagy in an mTOR-independent way ²⁴. The exogenously introduced calcium, in the form of CPP, would likely increase the intracellular cytosolic calcium. In addition, the transient nature of the induction may suggest that the level of cytosolic calcium eventually returns to the normal. How exactly CPP induces autophagy is currently under active investigation. Preliminary study suggested that CPP-induced GFP-LC3 punctation was not suppressed by STO-609, an inhibitor of CaMKKβ (data not shown), suggesting that other pathways may be involved. Whatever the potential signaling pathways may be, CPP proves to be a very reproducible and effective agent to induce autophagy, which constitutes an excellent model to study the mechanisms of autophagy induction, particularly those related to the effects of calcium, because the calcium could be introduced into cells without obvious prior disturbance of other cellular functions.

Materials and Methods

Antibodies and chemicals

The following antibodies were used: anti-GFP and anti-p62/SQSTM1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-Beclin-1 (BD Biosciences, San Diego, CA), and anti-β-actin (Sigma, St. Louis, MO). All other chemicals were from Sigma or Invitrogen (Carlsbad, CA).

Recombinant DNA constructs

pEGFP-LC3, expressing a fused protein of EGFP and human LC3B, was generated by inserting human LC3B open reading frame into pEGFP-C1 (Clontech Laboratories, In., Mountain View, CA). pGFP-LC3G120A was made by replacing Gly120 of LC3B with alanine. Similar method was used to generate the pRFP-LC3 and pRFP-LC3G120A using pDsRed/monomer C1 (Clontech). The tandem RFP-GFP-LC3 construct was reported previously ¹⁸. Specific siRNA against Beclin 1 and the control scrambled siRNA had been described before ²⁵.

Cell lines and transfection

HEK293, HCT116 (Bax −/−)²⁵, wild type and Atg5 −/−MEF ²⁶ were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum. Two stable cell lines, HEK293 and HCT116 (Bax−/−), expressing GFP-LC3 were established using retroviral vector carrying GFPLC3 fusion gene. 293 RFP-LC3, 293 RFP-GFP-LC3 and 293-GFP stably cell lines were established by transfection of plasmid DNA and selection under G418 (800 μg/ml). Adenoviral vector expressing GFP-LC3 gene was used for MEF cells transduction. RFP and mutant LC3 constructs were introduced into cells by transient transfection using Lipofectamine 2000 (Invitrogen). siRNAs were introduced into cells using Oligofectamine (Invitrogen). Cells were subjected to different treatments 48 hours post transfection except for adenoviral-mediated transduction, where subsequent treatment was given 16 hours later.

CPP treatment

Extracelluar calcium was introduced into cells in the form of calcium phosphate precipitates (CPP). CPP is freshly made by mixing an equal volume of 2x Buffer A (50 mM HEPES, pH7.05, 3 mM Na₂HPO₄) and 256 mM of calcium chloride solution. Cells were cultured in complete DMEM medium in 12-well plates at 2 × 10⁵ cells/per well. Fresh medium was exchanged 16 hours later. CPP mixture was added into individual wells in different amounts. Other treatments, if any, were given before or at the time of the CPP administration. At different time point, cells were observed or lysates were prepared.

Immunoblot and immunostaining analysis

These procedures have been described previously ²⁵. Briefly, lysates (10 μg) from cells were separated by SDS-PAGE and then transferred to PVDF membranes. Following the application of the primary antibodies and the HRP-conjugated secondary antibodies, the blots were developed using chemiluminescent substrates the signals were recorded using the Kodak Image Station 4000MM (Carestream Molecular Imaging, New Heaven, CT). For immunostaining, cells were first fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After wash, the cells were stained with a rabbit anti-LC3 antibody, followed by Cy-3 labeled anti-rabbit secondary antibody. Fluorescence images were acquired as descried in the section of fluorescence and electron microscopy.

Fluorescence and electron microscopy

For fluorescence microscopy, images were digitally acquired with a fluorescence microscope (Nikon TE200, Nikon, Melville, NY). For electron microscopy, cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 1 hour, and post-fixed in 1% osmium tetroxide in 0.1M cacodylate buffer for 1 hour. After dehydration and embedding, ultra thin sections were cut, stained with uranyl acetate and lead citrate and examined using a JEM1016CX transmission electron microscopy. Autophagic vesicles (AV), which include both autophagosomes and autolysosomes, were quantified and expressed as the number per given cell section.