Shang Xie1, Hui Xu1,2, Xiao-Feng Shan3, Zhi-Gang Cai3. 1. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital Stomatology, 22# Zhongguancun South Avenue, Haidian District, Beijing 100081, China. 2. Department of General Dentistry, Wang Fujing Branch, School of Stomatology, Capital Medical University, 11# Xila Hutong, Dongcheng District, Beijing 100006, China. 3. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital Stomatology, 22# Zhongguancun South Avenue, Haidian District, Beijing 100081, China c2013xs@163.com kqsxf@263.net.
Abstract
Botulinum toxin type A (BTXA) is a neurotoxic protein produced by Clostridium botulinum Our previous studies demonstrated that BTXA inhibits the secretory function of submandibular gland (SMG) and changes its structure. Several studies reported that SMG damage and repair often occur with autophagy in the rat. However, no studies reported whether secretory inhibition and structural changes of SMG after BTXA injection is related with autophagy. The present study was carried out to explore the association between BTXA injection and autophagy in rat SMG. Western blotting and immunofluorescence were used to detect the expression and distribution of light chain 3 (LC3) in rat SMG. MTS was used to detect the toxicity of BTXA on rat SMG-C6 cell line. GFP-LC3 and Lyso-Tracker Red fluorescence probe were used to assess the levels of autophagosomes and lysosome fusion and the effect of BTXA on autophagic flux in SMG-C6. Western blotting and immunofluorescence results showed that BTXA temporarily increased autophagosomes in rat SMG. MTS results showed that BTXA exerted its toxicity on SMG-C6 in a dose-dependent manner. BTXA increased the number of autophagosomes in SMG-C6; however, most autophagosomes did not colocalize with lysosome. Therefore, we presume that BTXA can change autophagic flux of SMG cells, the mechanism of which might relate with BTXA's disturbing autophagosome-lysosome fusion.
Botulinum toxin type A (BTXA) is a neurotoxic protein produced by Clostridium botulinum Our previous studies demonstrated that BTXA inhibits the secretory function of submandibular gland (SMG) and changes its structure. Several studies reported that SMG damage and repair often occur with autophagy in the rat. However, no studies reported whether secretory inhibition and structural changes of SMG after BTXA injection is related with autophagy. The present study was carried out to explore the association between BTXA injection and autophagy in rat SMG. Western blotting and immunofluorescence were used to detect the expression and distribution of light chain 3 (LC3) in rat SMG. MTS was used to detect the toxicity of BTXA on ratSMG-C6 cell line. GFP-LC3 and Lyso-Tracker Red fluorescence probe were used to assess the levels of autophagosomes and lysosome fusion and the effect of BTXA on autophagic flux in SMG-C6. Western blotting and immunofluorescence results showed that BTXA temporarily increased autophagosomes in rat SMG. MTS results showed that BTXA exerted its toxicity on SMG-C6 in a dose-dependent manner. BTXA increased the number of autophagosomes in SMG-C6; however, most autophagosomes did not colocalize with lysosome. Therefore, we presume that BTXA can change autophagic flux of SMG cells, the mechanism of which might relate with BTXA's disturbing autophagosome-lysosome fusion.
Botulinum toxin (BTX) is a neurotoxic protein produced by Clostridium botulinum [1-3]. Amongst the eight BTX types (A–H), Botulinum toxin type A (BTXA) has been widely used to treat excessive gland secretion, like hyperlacrimation, drooling, and Frey’s syndrome [4,5]. Our previous studies confirmed that BTXA can temporarily inhibit submandibular gland (SMG) secretion, a process that arose from BTXA-induced SMG cell apoptosis and aquaporin 5 (AQP5) expression and distribution [6-8]. However, the mechanism about how BTXA inhibits salivary secretion and changes the tissue structure of SMG remains unclear.Autophagy plays in the development of eukaryotic cells and related diseases [9-11]. It has four stages, namely the formation of a separation membrane, the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of autophagic products. The activation of autophagy requires many proteins, amongst which light chain 3 (LC3) is essential for autophagosome formation, and SQSTM1/p62 (sequestosome 1, p62) for autophagic substrate degradation. Autophagic flux is a process by which phagosomes fuse into lysosomes and degrade their contents [12,13]. This process occurs not when autophagy is going, but when autophagy is completed [12]. Autophagy is currently considered to be closely related to tumors, cardiovascular diseases, neurodegenerative diseases, aging, autoimmune diseases, and tissue fibrosis [10,14-19].In 2010, Silver et al. found that glandular injury repair was accompanied with activation of autophagy and mTOR signaling pathway [20]. Zoukhri et al. showed that IL-1β-induced repair of lacrimal gland inflammation was accompanied with autophagy in mice [21]. The above studies verified the association between glandular repair and autophagy. However, whether the inhibited salivary secretion and changed glandular structure after BTXA injection are related to autophagy remains unclear. To answer this question, the present study explored the associations between BTXA injection and autophagy and autophagic flux in rat SMG at tissue and cell levels.
Materials and methods
Experimental animals and BTXA injection
Healthy adult male SD rats (body weight 230–250 g) were purchased from the Experimental Animal Center of Peking University Health Science Center. All experimental procedures were approved by the Animal Research Ethics Committee (Peking University Health Science Center) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).For BTXA injection, 18 rats were randomly divided into six groups (three per group), including control group and five experimental groups (1 week group, 2 weeks group, 4 weeks group, 12 weeks group, and 24 weeks group). Under anesthesia, the rats were placed in a supine position and routinely sterilized. A midline incision was made in the neck to expose the bilateral submandibular glands. Each rat in the control group was injected with 0.1 ml of normal saline in the left submandibular gland. In order to study the effect of BTXA on the submandibular gland, each rat in the BTXA group was injected with three units BTXA (dissolved in 0.1 ml of normal saline). The submandibular gland specimens were collected under anesthesia at 1, 2, 4, 12, and 24 weeks after BTXA injection.
Reagents and antibodies
BTXA was gifted by Lanzhou Institute of Biological Products, China. One unit BTXA from this institute is equal to one unit of BOTOX (Allergan, Irvine, CA, U.S.A.) and 3–4 units of Dysport (Ipsen, Slough, U.K.). Antibodies for LC3-II and SQSTM1/p62 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). β-actin antibody and FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). CellTiter 96®AQueous One Solution cell proliferation assay (MTS assay) was purchased from Promega (Madison, WI, U.S.A.).
Cell culture and transfection
The rat submandibular gland SMG-C6 cell line was cultured in DMEM/F12 (1:1) as previous description [7,22]. The cells were cultured in a 37°C incubator containing 5% CO2 and transfected with cDNA of green fluorescent protein (GFP)–LC3 by use of MegeTran 1.0 (OriGene) according to the manufacturer’s instructions. After 24 h, the cells were kept in culture medium with 200 μg/ml G418 and maintained for 5 d.
Cell viability assay
The toxic effect of BTXA on SMG-C6 cell line was detected using MTS assay. The cells were seeded in 96-well plates (5000 cells per well, 100 μl per well), treated with various concentrations of BTXA (0, 50, 100, 200, and 300 U/ml), and maintained for 30 min, 24, 48, and 72 h, respectively. At each time point, 20 μl of MTS solution was added to each well and allowed to culture for 2–4 h. After incubating, the light absorption value of each well was measured at the wavelength of 490 nm on an enzyme-linked immunosorbent monitor.
Immunofluorescence and staining
Frozen sections (5-μm thick) of rat SMGs were fixed in cold acetone for 15 min, and immunostained with primary antibody against LC-3 (1:100) overnight at 4°C, then secondary antibodies (Invitrogen, CA21206s) for 2 h at RT. Nuclei were stained with 4′, 6-diamidino-2- phenylindole (DAPI) at RT for 10 min. Fluorescence images were captured by confocal microscope (TCSSP5; Leica, Germany) and typical images were presented.
Western blotting analysis
Total protein samples from rat SMG or SMG-C6 cell line were extracted in protein lysis buffer (in mM: 50 Tris-HCl, 5 EDTA, 5 EGTA, 150 NaCl, 40 NaF, 2.175 sodium orthovanadate, 1% TritonX-100, 0.1% SDS, 0.1% sodium deoxycholate, 0.1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2). Protein concentration was measured using BCA method. Equal amount of protein (25 μg) was separated by SDS/PAGE gel and transferred to PVDF membrane. The above membranes were blocked with 5% non-fat milk for 1 h, then incubated with primary antibodies overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase were incubated at RT for 2 h. Images were captured by ChemiDoc™XRS+ (Bio-Rad) with Image Lab software and quantitated by using Quantity One analysis software.
Autophagy flux assay
SMG-C6 cells stably expressing GFP-LC3 were cultured on confocal dishes. The cells were randomly divided into three groups: control group (cultured with DMEM/F12 with 10% FBS), BTXA treatment group (cultured with control group medium and treated with 100 unit/ml, BTXA), and fasting group (DMEM/F12 without FBS). After 24 h of treatment, the cells were incubated with Lyso-Tracker Red working solution and maintained in 37°C incubator for 30 min. The cells were then washed for three times with PBS that was then replaced with fresh cell culture solution. Images were captured by using confocal microscope (TCSSP5; Leica, Germany).
Statistical analysis
All experiments were performed in triplicate and repeated for three times using matched controls, and the data were pooled. The data were presented as mean ± S.E.M. One-way ANOVA analysis was performed to compare differences amongst groups. All statistical analyses were done using the GraphPad Prism 5.0 software under the Windows operating system. *P< 0.05 and **P< 0.01 were determined to be statistically significant.
Results
BTXA increased the expression and changed the distribution of autophagy-associated protein LC-3 in rat SMG
In the control group, immunofluorescence results showed that the LC-3 fluorescence was diffusely distributed in the cytoplasm of acinar cells. After BTXA injection for 1 and 2 weeks, LC-3 fluorescence was strongly spotted in the cytoplasm of acinar cells, and gradually recovered at 4 weeks. At 12 and 24 weeks, the spot-like structure disappeared and the fluorescent staining became diffusely distributed (Figure 1A). Western blotting results showed that LC3-II expression was increased at 1–2 weeks after BTXA injection and gradually declined during 4–24 weeks (Figure 1B). These results suggested that BTXA can temporarily increase the autophagosomes in rat SMG cells.
Figure 1
Effect of BTXA on LC3 expression and distribution in rat submandibular glands
(A) Immunofluorescence, scale bar, 25 μm; (B) results of western blotting. (control group, 1 week, 2 weeks, 4 weeks, 12 weeks, and 24 weeks).
Effect of BTXA on LC3 expression and distribution in rat submandibular glands
(A) Immunofluorescence, scale bar, 25 μm; (B) results of western blotting. (control group, 1 week, 2 weeks, 4 weeks, 12 weeks, and 24 weeks).
BTXA reduced the viability of SMG-C6 cells
In order to clarify the toxic effects of BTXA on SMG-C6 cells, MTS assay was used to observe the effect of BTXA (0, 50, 100, 200, and 300 U/ml) on the viability of SMG-C6 cells for 30 min, 24, 48, and 72 h, respectively. MTS results showed that BTXA reduced the viability of SMG-C6 cells in a dose-dependent (Figure 2). In order to clarify the effect of BTXA on SMG-C6 cells proliferation, we incubated SMG-C6 cells with BTXA for different duration time and monitored their cell proliferation activities. The results showed that the inhibitory effect of BTXA on SMG-C6 cells lasted approximately 24 h (Figure 3). And when the exposure time of BTXA was exposed to 5% CO2 at 37°C was for 48 h, the inhibitory function of BTXA on cells disappeared and the cells started to proliferate.
Figure 2
Effect of different concentrations of BTXA on SMG-C6 cells at specific times incubation time
Effect of BTXA on the expression of LC3 and p62 proteins in SMG-C6 cells
To examine the effect of BTXA on LC3 and p62 proteins in SMG-C6 cells, we used different concentrations of BTXA to culture the cells for 24 h. The results showed (Figure 4) that the expression level of LC3 II was significantly increased in BTXA medium at 50 and 100 U/ml for 24 h (n=3, P<0.05). Fasting group was used as positive control. The expression level of p62 protein was relatively higher than that in the control group, but the two groups showed no statistical difference (n=3, P>0.05).
Figure 4
Effect of different concentrations of BTXA on LC3 and p62 protein expression
Incubation concentrations of BTXA including 0 U/ml, 25 U/ml, 50 U/ml, 100 U/ml. (A) LC-3, N=3; (B) p62, N=3. Con (+): positive control group. Mean ± S.E., **P<0.01 and *P<0.05.
Effect of different concentrations of BTXA on LC3 and p62 protein expression
Incubation concentrations of BTXA including 0 U/ml, 25 U/ml, 50 U/ml, 100 U/ml. (A) LC-3, N=3; (B) p62, N=3. Con (+): positive control group. Mean ± S.E., **P<0.01 and *P<0.05.
BTXA interfered with the fusion of autophagosomes and lysosomes
In the absence of autophagy, the autophagosome marker GFP-LC3 fusion protein was diffusely distributed in the cell; when autophagy occurs, GFP-LC3 was transferred to the autophagosome membrane and appeared as bright green spots under fluorescence microscope. LysoTrackerRed is a red lysosomal fluorescent probe located on the lysosome. Under fluorescence microscopy, when autophagosomes and lysosomes did not fuse, the autophagosomes showed green fluorescent spots and lysosomes showed red fluorescent spots. After fusion, autophagosomes and lysosomes were combined. The results of immunofluorescence co-localization (Figure 5) showed that the green fluorescence was diffusely distributed in the normal control group, suggesting that fewer autophagosomes and no obvious yellow spots appeared when the two were co-localized. In the positive control group (starvation group), the green fluorescent spots significantly and the yellow spots co-localized with the lysosomes significantly increased compared with the control group. In the BTXA group, the green spots and red fluorescent spots significantly increased compared with the normal control group, indicating an increase in the number of autophagosomes and lysosomes; after co-localization, most of the green spots and red spots in the cells were separated, indicating that BTXA interfered with the fusion of autophagosomes with lysosomes.
Figure 5
Effect of BTXA on the autophagosome and lysosome fusion in SMG-C6
Discussion
Saliva is critical for maintaining an oral moisturization, digestion, and immunity. Our previous studies proved that BTXA decreased the salivation via inducing SMG cell apoptosis and changing AQP5 expression and distribution, but the hidden mechanism has not been fully illustrated. As we all know, autophagy and apoptosis are two cellular processes intertwined. Thus, we designed the present study to dig out this mechanism of autophagy.In the present study, we first demonstrated that BTXA injection temporarily increased autophagosomes in the rat SMG cells. Further, we used ratSMG-C6 cell line as a model to explore the effect of BTXA on autophagic flux and the possible mechanisms at the cellular level.In the cell experiments, we found that BTXA imposed its toxicity to SMG-C6 cells in a dose-dependent manner, which was consistent with the findings that high concentration of BTXA inhibited gland secretion. In order to clarify the effect of BTXA on protein expression in SMG-C6 cells, we examined the expression of autophagy-related proteins LC-3 in SMG-C6 cells. BTXA increased the conversion of LC3-I–LC3-II in a dose-dependent manner, suggesting that BTXA can increase autophagosomes in submandibular gland cells at the cellular level. However, there was no decrease in the expression of substrate protein p62 during autophagy, implying that BTXA can interfere with the degradation of autophagosomes (say, the fusion of autophagosomes and lysosomes is blocked). In order to clarify the possible causes of LC3-II increase, we performed a co-localization experiment using GFP-LC3 fusion protein and lysosomal tracer Lyso-Tracker. The results suggested that BTXA interrupted the fusion of autophagosome with lysosome.Numerous studies in recent years have shown that abnormal autophagy is closely related to fibrosis of tissues and organs [15,16,23-25]. Pulmonary fibrosis is a dynamic process in which autophagic flow is gradually obstructed. When only inflammation and no fibrosis appear in the lung tissue, autophagy gears into an active state and autophagosomes are formed to remove the therapeutic substances; when the lung tissue turns fibrotic, the autophagic flux is blocked and the autophagy is disruppted; in this condition, autophagosomes and autophagic lysosomes formed in cells cannot be cleared [23]. Similarly, in the present study, the number of autophagosomes increased significantly at 1 and 2 weeks after BTXA injection and gradually declined at 4–24 weeks, reflecting the changes in gland structures and functions. After BTXA injection at 1 and 2 weeks, the autophagic flux in SMG cells was blocked, salivary secretion was affected and the gland structure gradually changed.In summary, the present study found for the first time that BTXA can increase the number of autophagosomes in rat SMG and induce changes in autophagic flux in ratSMG-C6 cell line, and its potential mechanisms might be related with BTXA’s interference with autophagosme–lysosome fusion. These findings provided new insight into the mechanism of glandular secretion and structural change after BTXA injection.
Authors: Eri Iwai-Kanai; Hua Yuan; Chengqun Huang; M Richard Sayen; Cynthia N Perry-Garza; Lucy Kim; Roberta A Gottlieb Journal: Autophagy Date: 2008-01-18 Impact factor: 16.016
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Vladimir Kirkin; Lorrie A Kirshenbaum; Katsuhiko Kitamoto; Kaio Kitazato; Ludger Klein; Walter T Klimecki; Jochen Klucken; Erwin Knecht; Ben C B Ko; Jan C Koch; Hiroshi Koga; Jae-Young Koh; Young Ho Koh; Masato Koike; Masaaki Komatsu; Eiki Kominami; Hee Jeong Kong; Wei-Jia Kong; Viktor I Korolchuk; Yaichiro Kotake; Michael I Koukourakis; Juan B Kouri Flores; Attila L Kovács; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Carole Kretz-Remy; Anna M Krichevsky; Guido Kroemer; Rejko Krüger; Oleg Krut; Nicholas T Ktistakis; Chia-Yi Kuan; Roza Kucharczyk; Ashok Kumar; Raj Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Tino Kurz; Ho Jeong Kwon; Albert R La Spada; Frank Lafont; Trond Lamark; Jacques Landry; Jon D Lane; Pierre Lapaquette; Jocelyn F Laporte; Lajos László; Sergio Lavandero; Josée N Lavoie; Robert Layfield; Pedro A Lazo; Weidong Le; Laurent Le Cam; Daniel J Ledbetter; Alvin J X Lee; Byung-Wan Lee; Gyun Min Lee; Jongdae Lee; Ju-Hyun Lee; Michael Lee; Myung-Shik Lee; Sug Hyung Lee; Christiaan Leeuwenburgh; Patrick Legembre; Renaud Legouis; Michael Lehmann; Huan-Yao Lei; Qun-Ying Lei; David A Leib; José Leiro; John J Lemasters; Antoinette Lemoine; Maciej S Lesniak; Dina Lev; Victor V Levenson; Beth Levine; Efrat Levy; Faqiang Li; Jun-Lin Li; Lian Li; Sheng Li; Weijie Li; Xue-Jun Li; Yan-bo Li; Yi-Ping Li; Chengyu Liang; Qiangrong Liang; Yung-Feng Liao; Pawel P Liberski; Andrew Lieberman; Hyunjung J Lim; Kah-Leong Lim; Kyu Lim; Chiou-Feng Lin; Fu-Cheng Lin; Jian Lin; Jiandie D Lin; Kui Lin; Wan-Wan Lin; Weei-Chin Lin; Yi-Ling Lin; Rafael Linden; Paul Lingor; Jennifer Lippincott-Schwartz; Michael P Lisanti; Paloma B Liton; Bo Liu; Chun-Feng Liu; Kaiyu Liu; Leyuan Liu; Qiong A Liu; Wei Liu; Young-Chau Liu; Yule Liu; Richard A Lockshin; Chun-Nam Lok; Sagar Lonial; Benjamin Loos; Gabriel Lopez-Berestein; Carlos López-Otín; Laura Lossi; Michael T Lotze; Peter Lőw; Binfeng Lu; Bingwei Lu; Bo Lu; Zhen Lu; Frédéric Luciano; Nicholas W Lukacs; Anders H Lund; Melinda A Lynch-Day; Yong Ma; Fernando Macian; Jeff P MacKeigan; Kay F Macleod; Frank Madeo; Luigi Maiuri; Maria Chiara Maiuri; Davide Malagoli; May Christine V Malicdan; Walter Malorni; Na Man; Eva-Maria Mandelkow; Stéphen Manon; Irena Manov; Kai Mao; Xiang Mao; Zixu Mao; Philippe Marambaud; Daniela Marazziti; Yves L Marcel; Katie Marchbank; Piero Marchetti; Stefan J Marciniak; Mateus Marcondes; Mohsen Mardi; Gabriella Marfe; Guillermo Mariño; Maria Markaki; Mark R Marten; Seamus J Martin; Camille Martinand-Mari; Wim Martinet; Marta Martinez-Vicente; Matilde Masini; Paola Matarrese; Saburo Matsuo; Raffaele Matteoni; Andreas Mayer; Nathalie M Mazure; David J McConkey; Melanie J McConnell; Catherine McDermott; Christine McDonald; Gerald M McInerney; Sharon L McKenna; BethAnn McLaughlin; Pamela J McLean; Christopher R McMaster; G Angus McQuibban; Alfred J Meijer; Miriam H Meisler; Alicia Meléndez; Thomas J Melia; Gerry Melino; Maria A Mena; Javier A Menendez; Rubem F S Menna-Barreto; Manoj B Menon; Fiona M Menzies; Carol A Mercer; Adalberto Merighi; Diane E Merry; Stefania Meschini; Christian G Meyer; Thomas F Meyer; Chao-Yu Miao; Jun-Ying Miao; Paul A M Michels; Carine Michiels; Dalibor Mijaljica; Ana Milojkovic; Saverio Minucci; Clelia Miracco; Cindy K Miranti; Ioannis Mitroulis; Keisuke Miyazawa; Noboru Mizushima; Baharia Mograbi; Simin Mohseni; Xavier Molero; Bertrand Mollereau; Faustino Mollinedo; Takashi Momoi; Iryna Monastyrska; Martha M Monick; Mervyn J Monteiro; Michael N Moore; Rodrigo Mora; Kevin Moreau; Paula I Moreira; Yuji Moriyasu; Jorge Moscat; Serge Mostowy; Jeremy C Mottram; Tomasz Motyl; Charbel E-H Moussa; Sylke Müller; Sylviane Muller; Karl Münger; Christian Münz; Leon O Murphy; Maureen E Murphy; Antonio Musarò; Indira Mysorekar; Eiichiro Nagata; Kazuhiro Nagata; Aimable Nahimana; Usha Nair; Toshiyuki Nakagawa; Kiichi Nakahira; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Naweed I Naqvi; Derek P Narendra; Masashi Narita; Miguel Navarro; Steffan T Nawrocki; Taras Y Nazarko; Andriy Nemchenko; Mihai G Netea; Thomas P Neufeld; Paul A Ney; Ioannis P Nezis; Huu Phuc Nguyen; Daotai Nie; Ichizo Nishino; Corey Nislow; Ralph A Nixon; Takeshi Noda; Angelika A Noegel; Anna Nogalska; Satoru Noguchi; Lucia Notterpek; Ivana Novak; Tomoyoshi Nozaki; Nobuyuki Nukina; Thorsten Nürnberger; Beat Nyfeler; Keisuke Obara; Terry D Oberley; Salvatore Oddo; Michinaga Ogawa; Toya Ohashi; Koji Okamoto; Nancy L Oleinick; F Javier Oliver; Laura J Olsen; Stefan Olsson; Onya Opota; Timothy F Osborne; Gary K Ostrander; Kinya Otsu; Jing-hsiung James Ou; Mireille Ouimet; Michael Overholtzer; Bulent Ozpolat; Paolo Paganetti; Ugo Pagnini; Nicolas Pallet; Glen E Palmer; Camilla Palumbo; Tianhong Pan; Theocharis Panaretakis; Udai Bhan Pandey; Zuzana Papackova; Issidora Papassideri; Irmgard Paris; Junsoo Park; Ohkmae K Park; Jan B Parys; Katherine R Parzych; Susann Patschan; Cam Patterson; Sophie Pattingre; John M Pawelek; Jianxin Peng; David H Perlmutter; Ida Perrotta; George Perry; Shazib Pervaiz; Matthias Peter; Godefridus J Peters; Morten Petersen; Goran Petrovski; James M Phang; Mauro Piacentini; Philippe Pierre; Valérie Pierrefite-Carle; Gérard Pierron; Ronit Pinkas-Kramarski; Antonio Piras; Natik Piri; Leonidas C Platanias; Stefanie Pöggeler; Marc Poirot; Angelo Poletti; Christian Poüs; Mercedes Pozuelo-Rubio; Mette Prætorius-Ibba; Anil Prasad; Mark Prescott; Muriel Priault; Nathalie Produit-Zengaffinen; Ann Progulske-Fox; Tassula Proikas-Cezanne; Serge Przedborski; Karin Przyklenk; Rosa Puertollano; Julien Puyal; Shu-Bing Qian; Liang Qin; Zheng-Hong Qin; Susan E Quaggin; Nina Raben; Hannah Rabinowich; Simon W Rabkin; Irfan Rahman; Abdelhaq Rami; Georg Ramm; Glenn Randall; Felix Randow; V Ashutosh Rao; Jeffrey C Rathmell; Brinda Ravikumar; Swapan K Ray; Bruce H Reed; John C Reed; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; John J Reiners; Russel J Reiter; Jun Ren; José L Revuelta; Christopher J Rhodes; Konstantinos Ritis; Elizete Rizzo; Jeffrey Robbins; Michel Roberge; Hernan Roca; Maria C Roccheri; Stephane Rocchi; H Peter Rodemann; Santiago Rodríguez de Córdoba; Bärbel Rohrer; Igor B Roninson; Kirill Rosen; Magdalena M Rost-Roszkowska; Mustapha Rouis; Kasper M A Rouschop; Francesca Rovetta; Brian P Rubin; David C Rubinsztein; Klaus Ruckdeschel; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Nelson Ruiz-Opazo; Rossella Russo; Tor Erik Rusten; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Junichi Sadoshima; Tapas Saha; Tatsuya Saitoh; Hiroshi Sakagami; Yasuyoshi Sakai; Ghasem Hoseini Salekdeh; Paolo Salomoni; Paul M Salvaterra; Guy Salvesen; Rosa Salvioli; Anthony M J Sanchez; José A Sánchez-Alcázar; Ricardo Sánchez-Prieto; Marco Sandri; Uma Sankar; Poonam Sansanwal; Laura Santambrogio; Shweta Saran; Sovan Sarkar; Minnie Sarwal; Chihiro Sasakawa; Ausra Sasnauskiene; Miklós Sass; Ken Sato; Miyuki Sato; Anthony H V Schapira; Michael Scharl; Hermann M Schätzl; Wiep Scheper; Stefano Schiaffino; Claudio Schneider; Marion E Schneider; Regine Schneider-Stock; Patricia V Schoenlein; Daniel F Schorderet; Christoph Schüller; Gary K Schwartz; Luca Scorrano; Linda Sealy; Per O Seglen; Juan Segura-Aguilar; Iban Seiliez; Oleksandr Seleverstov; Christian Sell; Jong Bok Seo; Duska Separovic; Vijayasaradhi Setaluri; Takao Setoguchi; Carmine Settembre; John J Shacka; Mala Shanmugam; Irving M Shapiro; Eitan Shaulian; Reuben J Shaw; James H Shelhamer; Han-Ming Shen; Wei-Chiang Shen; Zu-Hang Sheng; Yang Shi; Kenichi Shibuya; Yoshihiro Shidoji; Jeng-Jer Shieh; Chwen-Ming Shih; Yohta Shimada; Shigeomi Shimizu; Takahiro Shintani; Orian S Shirihai; Gordon C Shore; Andriy A Sibirny; Stan B Sidhu; Beata Sikorska; Elaine C M Silva-Zacarin; Alison Simmons; Anna Katharina Simon; Hans-Uwe Simon; Cristiano Simone; Anne Simonsen; David A Sinclair; Rajat Singh; Debasish Sinha; Frank A Sinicrope; Agnieszka Sirko; Parco M Siu; Efthimios Sivridis; Vojtech Skop; Vladimir P Skulachev; Ruth S Slack; Soraya S Smaili; Duncan R Smith; Maria S Soengas; Thierry Soldati; Xueqin Song; Anil K Sood; Tuck Wah Soong; Federica Sotgia; Stephen A Spector; Claudia D Spies; Wolfdieter Springer; Srinivasa M Srinivasula; Leonidas Stefanis; Joan S Steffan; Ruediger Stendel; Harald Stenmark; Anastasis Stephanou; Stephan T Stern; Cinthya Sternberg; Björn Stork; Peter Strålfors; Carlos S Subauste; Xinbing Sui; David Sulzer; Jiaren Sun; Shi-Yong Sun; Zhi-Jun Sun; Joseph J Y Sung; Kuninori Suzuki; Toshihiko Suzuki; Michele S Swanson; Charles Swanton; Sean T Sweeney; Lai-King Sy; Gyorgy Szabadkai; Ira Tabas; Heinrich Taegtmeyer; Marco Tafani; Krisztina Takács-Vellai; Yoshitaka Takano; Kaoru Takegawa; Genzou Takemura; Fumihiko Takeshita; Nicholas J Talbot; Kevin S W Tan; Keiji Tanaka; Kozo Tanaka; Daolin Tang; Dingzhong Tang; Isei Tanida; Bakhos A Tannous; Nektarios Tavernarakis; Graham S Taylor; Gregory A Taylor; J Paul Taylor; Lance S Terada; Alexei Terman; Gianluca Tettamanti; Karin Thevissen; Craig B Thompson; Andrew Thorburn; Michael Thumm; FengFeng Tian; Yuan Tian; Glauco Tocchini-Valentini; Aviva M Tolkovsky; Yasuhiko Tomino; Lars Tönges; Sharon A Tooze; Cathy Tournier; John Tower; Roberto Towns; Vladimir Trajkovic; Leonardo H Travassos; Ting-Fen Tsai; Mario P Tschan; Takeshi Tsubata; Allan Tsung; Boris Turk; Lorianne S Turner; Suresh C Tyagi; Yasuo Uchiyama; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Vivek K Unni; Maria I Vaccaro; Enza Maria Valente; Greet Van den Berghe; Ida J van der Klei; Wouter van Doorn; Linda F van Dyk; Marjolein van Egmond; Leo A van Grunsven; Peter Vandenabeele; Wim P Vandenberghe; Ilse Vanhorebeek; Eva C Vaquero; Guillermo Velasco; Tibor Vellai; Jose Miguel Vicencio; Richard D Vierstra; Miquel Vila; Cécile Vindis; Giampietro Viola; Maria Teresa Viscomi; Olga V Voitsekhovskaja; Clarissa von Haefen; Marcela Votruba; Keiji Wada; Richard Wade-Martins; Cheryl L Walker; Craig M Walsh; Jochen Walter; Xiang-Bo Wan; Aimin Wang; Chenguang Wang; Dawei Wang; Fan Wang; Fen Wang; Guanghui Wang; Haichao Wang; Hong-Gang Wang; Horng-Dar Wang; Jin Wang; Ke Wang; Mei Wang; Richard C Wang; Xinglong Wang; Xuejun Wang; Ying-Jan Wang; Yipeng Wang; Zhen Wang; Zhigang Charles Wang; Zhinong Wang; Derick G Wansink; Diane M Ward; Hirotaka Watada; Sarah L Waters; Paul Webster; Lixin Wei; Conrad C Weihl; William A Weiss; Scott M Welford; Long-Ping Wen; Caroline A Whitehouse; J Lindsay Whitton; Alexander J Whitworth; Tom Wileman; John W Wiley; Simon Wilkinson; Dieter Willbold; Roger L Williams; Peter R Williamson; Bradly G Wouters; Chenghan Wu; Dao-Cheng Wu; William K K Wu; Andreas Wyttenbach; Ramnik J Xavier; Zhijun Xi; Pu Xia; Gengfu Xiao; Zhiping Xie; Zhonglin Xie; Da-zhi Xu; Jianzhen Xu; Liang Xu; Xiaolei Xu; Ai Yamamoto; Akitsugu Yamamoto; Shunhei Yamashina; Michiaki Yamashita; Xianghua Yan; Mitsuhiro Yanagida; Dun-Sheng Yang; Elizabeth Yang; Jin-Ming Yang; Shi Yu Yang; Wannian Yang; Wei Yuan Yang; Zhifen Yang; Meng-Chao Yao; Tso-Pang Yao; Behzad Yeganeh; Wei-Lien Yen; Jia-jing Yin; Xiao-Ming Yin; Ook-Joon Yoo; Gyesoon Yoon; Seung-Yong Yoon; Tomohiro Yorimitsu; Yuko Yoshikawa; Tamotsu Yoshimori; Kohki Yoshimoto; Ho Jin You; Richard J Youle; Anas Younes; Li Yu; Long Yu; Seong-Woon Yu; Wai Haung Yu; Zhi-Min Yuan; Zhenyu Yue; Cheol-Heui Yun; Michisuke Yuzaki; Olga Zabirnyk; Elaine Silva-Zacarin; David Zacks; Eldad Zacksenhaus; Nadia Zaffaroni; Zahra Zakeri; Herbert J Zeh; Scott O Zeitlin; Hong Zhang; Hui-Ling Zhang; Jianhua Zhang; Jing-Pu Zhang; Lin Zhang; Long Zhang; Ming-Yong Zhang; Xu Dong Zhang; Mantong Zhao; Yi-Fang Zhao; Ying Zhao; Zhizhuang J Zhao; Xiaoxiang Zheng; Boris Zhivotovsky; Qing Zhong; Cong-Zhao Zhou; Changlian Zhu; Wei-Guo Zhu; Xiao-Feng Zhu; Xiongwei Zhu; Yuangang Zhu; Teresa Zoladek; Wei-Xing Zong; Antonio Zorzano; Jürgen Zschocke; Brian Zuckerbraun Journal: Autophagy Date: 2012-04 Impact factor: 16.016
Authors: Avignat S Patel; Ling Lin; Alexander Geyer; Jeffrey A Haspel; Chang Hyeok An; Jiaofei Cao; Ivan O Rosas; Danielle Morse Journal: PLoS One Date: 2012-07-18 Impact factor: 3.240
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