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• W2066382194 abstract "Macroautophagy, a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles, is believed to be essential for cell survival or death in response to stress conditions. Recent observations indicate that autophagy is an adaptive response in cells subjected to prolonged hypoxia. However, the signaling mechanisms that activate autophagy under acute hypoxic stress are not clearly understood. In this study, we show that acute hypoxic stress by treatment with 1% O2 or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase C; (PKC;), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3). Interestingly, hypoxic stress leads to a rapid and transient activation of JNK in Pa-4 or mouse embryo fibroblast cells. Acute hypoxic stress-induced changes in LC3-II level and JNK activation are attenuated in Pa-4 cells by dominant negative PKC;KD or in mouse embryo fibroblast/PKC;-null cells. Intriguingly, the requirement of PKC; is not apparent for starvation-induced autophagy. The importance of PKC; in hypoxic stress-induced adaptive responses is further supported by our findings that inhibition of PKC;-facilitated autophagy by 3-methyladenine or Atg5 knock-out renders a greater prevalence of cell death following prolonged desferroxamine treatment, whereas PKC;- or JNK1-deficient cells exhibit resistance to extended hypoxic exposure. These results uncover dual roles of PKC;-dependent signaling in the cell fate determination upon hypoxic exposure. Macroautophagy, a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles, is believed to be essential for cell survival or death in response to stress conditions. Recent observations indicate that autophagy is an adaptive response in cells subjected to prolonged hypoxia. However, the signaling mechanisms that activate autophagy under acute hypoxic stress are not clearly understood. In this study, we show that acute hypoxic stress by treatment with 1% O2 or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase C; (PKC;), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3). Interestingly, hypoxic stress leads to a rapid and transient activation of JNK in Pa-4 or mouse embryo fibroblast cells. Acute hypoxic stress-induced changes in LC3-II level and JNK activation are attenuated in Pa-4 cells by dominant negative PKC;KD or in mouse embryo fibroblast/PKC;-null cells. Intriguingly, the requirement of PKC; is not apparent for starvation-induced autophagy. The importance of PKC; in hypoxic stress-induced adaptive responses is further supported by our findings that inhibition of PKC;-facilitated autophagy by 3-methyladenine or Atg5 knock-out renders a greater prevalence of cell death following prolonged desferroxamine treatment, whereas PKC;- or JNK1-deficient cells exhibit resistance to extended hypoxic exposure. These results uncover dual roles of PKC;-dependent signaling in the cell fate determination upon hypoxic exposure. Macroautophagy (hereafter referred to as autophagy), self-cannibalization to degrade cells' own constituents, including their organelles, is induced by certain environmental cues, such as starvation, heat shock, and hypoxia (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar). In mammals, autophagy exhibits marked associations with neurodegenerative diseases, cancer, cardiomyopathies, aging, type II programmed cell death, bacterial invasion, major histocompatibility complex class II antigen presentation, and other cellular maintenance (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar). The autophagy-related genes, atg/apg/aut/cvt genes, have been isolated and characterized in yeast and mammals (2Mizushima N. Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2954) Google Scholar). There are two ubiquitination-like conjugation systems required for autophagosome formation (reviewed in Refs. 1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar, 2Mizushima N. Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2954) Google Scholar, 3Mizushima N. Klionsky D.J. Annu. Rev. Nutr. 2007; 27: 19-40Crossref PubMed Scopus (632) Google Scholar). One system mediates the conjugation of Atg12-Atg5, whereas the other system produces covalent linkage between Atg8 and phosphatidylethanolamine. Atg12 is first activated by Atg7, followed by transfer to Atg10, and finally covalently attached to Atg5, a process requiring ATP (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar, 2Mizushima N. Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2954) Google Scholar, 3Mizushima N. Klionsky D.J. Annu. Rev. Nutr. 2007; 27: 19-40Crossref PubMed Scopus (632) Google Scholar). The Atg12-Atg5 conjugates localize to autophagosome precursors, dissociate just before or after completion of autophagic vacuole formation, and are essential for elongation of the isolation membrane (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar, 2Mizushima N. Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2954) Google Scholar, 3Mizushima N. Klionsky D.J. Annu. Rev. Nutr. 2007; 27: 19-40Crossref PubMed Scopus (632) Google Scholar). LC3 is the mammalian homologue of yeast Atg8 (4Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5466) Google Scholar). The carboxyl-terminal region of LC3 is cleaved by Atg4, generating a soluble form known as LC3-I and exposing carboxyl-terminal glycines essential for further reactions (5Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1116) Google Scholar). LC3-I, in turn, is modified to a membrane-bound form of LC3-II (a LC3-phospholipid conjugate) by Atg7 and Atg3, E1- 2The abbreviations used are: E1, ubiquitin-activating enzyme, E2, ubiquitin carrier protein; PKC;, protein kinase C;; DFO, desferroxamine; 3-MA, 3-methyladenine; CQ, chloroquine; EBSS, Earle's balanced salt solution without phenol red; PI3K, phosphoinositide 3-kinase; AMPK, AMP-activated protein kinase; MTOR, mammalian target of rapamycin; WT, wild type; MEF, mouse embryo fibroblast(s); JNK, Jun N-terminal kinase; JNKi, JNK peptide inhibitor; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; ER, endoplasmic reticulum. and E2-like enzymes (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar, 2Mizushima N. Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2954) Google Scholar, 3Mizushima N. Klionsky D.J. Annu. Rev. Nutr. 2007; 27: 19-40Crossref PubMed Scopus (632) Google Scholar), and localizes to autophagosomes and autolysosomes (4Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5466) Google Scholar). Thus, the relative amount of LC3-I-to-LC3-II conversion and the changes in LC3-II level via degradation in autolysosomes of mammalian cells are a useful marker for the formation of autophagosomes and autolysosomes, respectively. Importantly, LC3-II lipidation depends on the Atg12-Atg5 conjugates, since the LC3-II form is not observed at all in either Atg5-/- cells or those cells engineered to express the conjugation-defective Atg5 mutant (Atg5K130R) (6Hanada T. Noda N.N. Satomi Y. Ichimura Y. Fujioka Y. Takao T. Inagaki F. Ohsumi Y. J. Biol. Chem. 2007; 282: 37298-37302Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 7Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Signaling pathways that regulate autophagy are extremely complex, since numerous feedforward and feedback loops and cross-talks with many other signaling networks are involved in coordinating cellular autophagy, proliferation, and apoptosis. One of the key regulators of autophagy is PI3K. Mammalian cells contain three distinct types of PI3K, based on their substrate specificities and subunit organizations (8Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1372) Google Scholar). Class I PI3Ks produce phosphatidylinositol 3,4,5-trisphosphate in vivo. Class II PI3Ks appear to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate. Finally, the class III PI3K, Vps34, only produces phosphatidylinositol 3-phosphate. Whereas the class I PI3K activates the mammalian target of rapamycin (mTOR), the interruption of mTOR-dependent signaling by rapamycin stimulates autophagy in many cell types (9Rubinsztein D.C. Gestwicki J.E. Murphy L.O. Klionsky D.J. Nat. Rev. Drug Discov. 2007; 6: 304-312Crossref PubMed Scopus (890) Google Scholar). By contrast, the pharmacological inhibitor, 3-methyladenine (3-MA), which targets the class III PI3K/Vps34 inhibits the nucleation of autophagosome vesicles (10Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1035) Google Scholar). Although autophagy has been characterized in many contexts, the signaling pathway that activates autophagy in response to hypoxic stress has not yet been studied extensively. Oxygen deprivation by chronic hypoxia is known to induce autophagy. For example, Beclin-1 levels in the cortex and striatum are increased in cerebral ischemia (11Adhami F. Liao G. Morozov Y.M. Schloemer A. Schmithorst V.J. Lorenz J.N. Dunn R.S. Vorhees C.V. Wills-Karp M. Degen J.L. Davis R.J. Mizushima N. Rakic P. Dardzinski B.J. Holland S.K. Sharp F.R. Kuan C.Y. Am J. Pathol. 2006; 169: 566-583Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The early changes of Beclin-1 levels in the penumbra occurred at 6 h, peaked at 24 h, and lasted for at least 2 days in the neuronal cells (12Rami A. Autophagy. 2008; 4: 227-229Crossref PubMed Scopus (31) Google Scholar). Beclin-1 (also called Atg6) is a phylogenetically conserved protein that is essential for the initiation of autophagy, presumably via its interaction with the class III PI3K/Vps34 (1Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5587) Google Scholar, 13Zeng X. Overmeyer J.H. Maltese W.A. J. Cell Sci. 2006; 119: 259-270Crossref PubMed Scopus (291) Google Scholar). Originally, human Beclin-1 was identified as an interactor of Bcl-2 (14Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2751) Google Scholar). Beclin-1 reportedly possesses a so-called Bcl-2 homology region-3 (BH3) domain (amino acids 114-123) that mediates the interaction with Bcl-2 and other Bcl-2 homologues, such as Bcl-XL and Mcl-1 (15Maiuri M.C. Le Toumelin G. Criollo A. Rain J.C. Gautier F. Juin P. Tasdemir E. Pierron G. Troulinaki K. Tavernarakis N. Hickman J.A. Geneste O. Kroemer G. EMBO J. 2007; 26: 2527-2539Crossref PubMed Scopus (932) Google Scholar). It has recently become clear that overexpression of Beclin-1 or depletion of Bcl-2 stimulates autophagy (16Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell. 2005; 122: 927-939Abstract Full Text Full Text PDF PubMed Scopus (2955) Google Scholar). Herein, we demonstrate a novel pathway by which acute hypoxic stress utilizes a rapid activation of the PKC; signaling pathway to release Beclin-1 from Bcl-2, leading to autophagy induction. Cell Lines and Chemicals—Rat parotid epithelial cell lines, Pa-4 and Pa-4/PKC;KD, were generated and cultured as previously described (17Clavijo C. Chen J.L. Kim K.J. Reyland M.E. Ann D.K. Am. J. Physiol. 2007; 292: C2150-C2160Crossref Scopus (14) Google Scholar). MEF/WT, MEF/JNK1-/-, MEF/JNK2-/-, MEF/Atg5-/-, MEF/GFP-LC3, and MEF/PKC;-/- cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus 1% penicillin/streptomycin and grown at 37 °C. MCF-7/Neo and MCF-7/caspase-3 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 0.5 mg/ml Geneticin (G418; Sigma), and antibiotics. Desferroxamine (DFO), 3-methyladenine (3-MA), chloroquine (CQ), rapamycin, E64d, pepstatin A, Earle's balanced salt solution without phenol red (EBSS), Bafilomycin A1, and SP 600125 were purchased from Sigma. The cell membrane-permeable JNK peptide inhibitor (JNKi) harbors amino acids 48-60 of human immunodeficiency virus Tat protein and amino acids 32-50 of c-Jun. Myc-Bcl-2 Wild Type and Myc-Bcl2-S70A Mutant—Human Bcl-2 cDNA was reverse transcribed from total RNA with the gene-specific primer, 5′-GGTACATCACTGACAATGCA-3′. Subsequently, the Bcl-2-encoding fragment, tagged with appropriate restriction enzyme sites, was amplified from the cDNA by PCR and inserted between EcoRI and HindIII sites of expression vector pCMV-Tag3A (Stratagene). The S70A mutant was generated using the Transformer site-directed mutagenesis kit (Clontech) and the mutagenic primer, 5′-CGCCAGGACCGCGCCGCTGCAG-3′. DNA sequences of these constructs were verified by sequencing reactions. GFP-LC3 Puncta Analyses—Pa-4, MCF-7/caspase-3, or MCF-7/Neo cells were transfected with GFP-LC3 or GFP-N1 expression constructs by using Lipofectamine 2000 (Invitrogen) per the manufacturer's instructions. For GFP-LC3 puncta analyses, cells at 36 h post-transfection or stably transfected MEF/GFP-LC3 cells were seeded on coverslips placed in the 6-well plates and cultured overnight, followed by various treatments for different time periods. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, incubated with 4′-6-diamidino-2-phenylindole (Molecular Probes) for 10 min and mounted with the Prolong Gold Antifade reagent (Molecular Probes). Images were acquired with a Zeiss inverted LSM 510 Meta 2 photon microscope and analyzed using a Zeiss LSM image examiner. The GFP-LC3 punctae and GFP-LC3-positive cells were examined and quantified in more than five fields per slide on five slides. The increase in GFP-LC3 punctae represents the relative accumulation of autophagosomes. Measurement of GFP-LC3 Intensity by Fluorescence-activated Cell Sorter (FACS) Analysis—MEF cells that stably expressed GFP-LC3 were seeded subconfluently on the day before treatment in 6-well plates. Following the indicated treatment periods, cells were harvested with trypsin/EDTA, washed with PBS, and fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. Fixed cells were washed with PBS twice prior to subjecting them to FACS. Analysis of 1 × 105 cells/sample was performed by a CyAn™ ADP 9 Color Flow cytometer (DAKO; Analytic Cytometry Core Facility in City of Hope Medical Center), and viable cell counts were plotted as GFP fluorescence intensity by FlowJo Software (Tree Star, Inc., Ashland, OR). The level of GFP fluorescence intensity in each treated sample was normalized to the level of resting, vehicle-treated controls set at 100%. The relative level of GFP-LC3 intensity in each treatment was calculated from at least three independent experiments and represents mean ± S.D. in the graphs. Whole Cell Lysate Preparation and Western Analyses—For protein phosphorylation studies, whole cell lysates were extracted by SDS lysis buffer, as previously described (17Clavijo C. Chen J.L. Kim K.J. Reyland M.E. Ann D.K. Am. J. Physiol. 2007; 292: C2150-C2160Crossref Scopus (14) Google Scholar), containing both the Complete protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitor Na3VO4 (2 mm; Sigma) and subjected to SDS-PAGE and subsequently immunoblotted with respective antibodies for BNIP3 (Abcam), Bcl-2, or c-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-SAPK/JNK (Thr-183/Tyr-185), SAPK/JNK, phospho-c-Jun (Ser-63), c-Jun, phospho-Bcl-2 (Ser-70), phospho-AMPK; (Thr-172), or AMPK; (Cell Signaling Technology). Anti-PKC; and anti-GFP antibodies were purchased from Santa Cruz Biotechnologies. A mouse anti-Beclin-1 antibody was purchased from BD Biosciences. A mouse anti-phosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology. To detect endogenous LC3-I and LC3-II, whole cell lysates were prepared using a Triton X-100-based lysis buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100) plus the Complete protease inhibitor mixture (Roche Applied Science). Equal amounts of proteins were aliquoted and added with SDS-PAGE loading buffer immediately before boiling, followed by SDS-PAGE and subsequent immunoblotting with an anti-LC3 antibody (MBL International Co.). Anti-tubulin (Santa Cruz Biotechnology) and anti-actin (Chemicon International) antibodies were used to assess equal protein loading. Immunoblots using an enhanced chemiluminescence detection kit (ECL-Plus; Amersham Biosciences) were imaged with the VersaDoc 5000 Imaging System (Bio-Rad). Densitometric data were captured, quantitated with Quantity One Software (Bio-Rad), and normalized with internal control proteins, individually, in each experiment. The relative level of a particular protein altered by various treatments was then calculated by setting the normalized value in the control as 1, assuming equal variances. Immunoprecipitation—Whole cell lysates were prepared using radioimmune precipitation buffer (25 mm Tris, 125 mm NaCl, 1% Nonidet P-40 (Nonidet P-40), 0.1% SDS, 0.5% sodium deoxycholate, 0.004% sodium azide, pH 8.0) containing both the Complete protease inhibitor mixture and PhosSTOP phosphatase inhibitor mixture (Roche Applied Science). Protein concentrations were determined by a BCA assay (Bio-Rad). Whole cell lysates (1 mg) were incubated with 1-5 ;g/ml of a specific antibody for Beclin-1 (Abcam), PKC;, or c-Myc at 4 °C for 2 h, followed by incubation with 20 ;l of Protein A/G PLUS-agarose beads (Santa Cruz Biotechnologies) at 4 °C overnight to capture immune complexes. Immune complexes were then washed three times with PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and the Complete protease inhibitor mixture, dissolved by adding SDS-PAGE loading buffer, boiled for 6 min, resolved by SDS-PAGE, transferred to membranes, and subsequently blotted with appropriate antibodies. The levels for proteins of interest were determined using an ECL-Plus kit. The relative level of immunoprecipitated complex was normalized with total input proteins in lysates individually and normalized by setting the nontreatment sample value as 1. Cell Viability Assay—Cell viabilities were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Pa-4, MEF/WT and various knock-out cell lines were seeded into 24-well plates to reach 35% confluence on the day of treatments. The cells were treated with different regimens or vehicle as indicated for 0, 24, and 48 h, followed by the 3--(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) assay according to the manufacturer's recommendation. Absorbance of each well was read at 540 nm in a scanning multiwell spectrophotometer. The results were depicted as percentage for cell viability and reported as the mean ± S.D. of three independent experiments performed in triplicate. Statistical Analyses—Experiments were independently carried out at least three times, and one representative data set of the three independent experiments was presented where appropriate. The results were evaluated for statistical significance by two-way analyses of variance with randomized sample blocks, followed by post hoc comparisons based on Fisher's least squared difference method of protected t-tests. The error bars were marked as the S.D. of the mean. p values less than 0.05 were regarded as significant. Increased Autophagosome Accumulation during Acute Hypoxic Stress—We previously reported that treatment of cells with DFO, a hypoxia-mimetic agent, confers a cross-talk between the activation of PKC; and apoptotic caspase-3 pathways (17Clavijo C. Chen J.L. Kim K.J. Reyland M.E. Ann D.K. Am. J. Physiol. 2007; 292: C2150-C2160Crossref Scopus (14) Google Scholar). Since signaling pathways that regulate cell proliferation and apoptosis are frequently associated with the regulation of autophagy, we hypothesized that hypoxia or DFO treatment induces autophagy. To test this hypothesis, induction of autophagy detectable by Western analyses on the time course of the changes in the amount of membrane-bound LC3-II (the phosphatidylethanolamine-conjugated form (5Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1116) Google Scholar) and by GFP-LC3 puncta accumulation (reviewed in Ref. 18Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clave C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. DineshKumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Droge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fesus L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. Gonzalez-Estevez C. Gorski S. Gottlieb R.A. Haussinger D. He Y.W. Heidenreich K. Hill J.A. Hoyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jaattela M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovacs A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. Lopez-Otin C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Melendez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Munz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nurnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F. Rohrer B. Rubinsztein D.C. Ryan K.M. Sadoshima J. Sakagami H. Sakai Y. Sandri M. Sasakawa C. Sass M. Schneider C. Seglen P.O. Seleverstov O. Settleman J. Shacka J.J. Shapiro I.M. Sibirny A. SilvaZacarin E.C. Simon H.U. Simone C. Simonsen A. Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Talloczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcategui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1975) Google Scholar) was investigated. In Pa-4 cells, endogenous LC3 is mainly presented as the lipidated form of LC3-II in the normoxic and nutrient-rich condition (Fig. 1A, top, lane 1), similarly reported in A431, HeLa, and MDA-MB-231 cells (19Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (940) Google Scholar). First, we monitored the time course of the changes in LC3-II level as a means to assess autophagy progression, since it was reported that LC3-II level is decreased inside autolysosomes during the autophagic process (18Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clave C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. DineshKumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Droge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fesus L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. Gonzalez-Estevez C. Gorski S. Gottlieb R.A. Haussinger D. He Y.W. Heidenreich K. Hill J.A. Hoyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jaattela M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovacs A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. Lopez-Otin C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Melendez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Munz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nurnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F. Rohrer B. Rubinsztein D.C. Ryan K.M. Sadoshima J. Sakagami H. Sakai Y. Sandri M. Sasakawa C. Sass M. Schneider C. Seglen P.O. Seleverstov O. Settleman J. Shacka J.J. Shapiro I.M. Sibirny A. SilvaZacarin E.C. Simon H.U. Simone C. Simonsen A. Smith M.A. Spanel-Borowski K. Srinivas V. 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