Endogenous HMGB1 regulates autophagy.
ABSTRACT Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.
- SourceAvailable from: Dmitri V. Krysko[show abstract] [hide abstract]
ABSTRACT: A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called 'damage-associated molecular patterns' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.Cell Death & Disease 01/2013; 4:e631. · 6.04 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Autophagy is a fundamental eukaryotic process with multiple cytoplasmic homeostatic roles, recently expanded to include unique stand-alone immunological functions and interactions with nearly all parts of the immune system. In this article, we review this growing repertoire of autophagy roles in innate and adaptive immunity and inflammation. Its unique functions include cell-autonomous elimination of intracellular microbes facilitated by specific receptors. Other intersections of autophagy with immune processes encompass effects on inflammasome activation and secretion of its substrates, including IL-1β, effector and regulatory interactions with TLRs and Nod-like receptors, Ag presentation, naive T cell repertoire selection, and mature T cell development and homeostasis. Genome-wide association studies in human populations strongly implicate autophagy in chronic inflammatory disease and autoimmune disorders. Collectively, the unique features of autophagy as an immunological process and its contributions to other arms of the immune system represent a new immunological paradigm.The Journal of Immunology 07/2012; 189(1):15-20. · 5.52 Impact Factor
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ABSTRACT: High-mobility group box 1 protein (HMGB1), which mainly exists in the nucleus, has recently been shown to function as a sentinel molecule for viral nucleic acid sensing and an autophagy regulator in the cytoplasm. In this study, we studied the chaperone-like activity of HMGB1 and found that HMGB1 inhibited the chemically induced aggregation of insulin and lysozyme, as well as the heat-induced aggregation of citrate synthase. HMGB1 also restored the heat-induced suppression of cytoplasmic luciferase activity as a reporter protein in hamster lung fibroblast O23 cells with expression of HMGB1. Next, we demonstrated that HMGB1 inhibited the formation of aggregates and toxicity caused by expanded polyglutamine (polyQ), one of the main causes of Huntington disease. HMGB1 directly interacted with polyQ on immunofluorescence and coimmunoprecipitation assay, whereas the overexpression of HMGB1 or exogenous administration of recombinant HMGB1 protein remarkably reduced polyQ aggregates in SHSY5Y cells and hmgb1(-/-) mouse embryonic fibroblasts upon filter trap and immunofluorescence assay. Finally, overexpressed HMGB1 proteins in mouse embryonic primary striatal neurons also bound to polyQ and decreased the formation of polyQ aggregates. To this end, we have demonstrated that HMGB1 exhibits chaperone-like activity and a possible therapeutic candidate in polyQ disease.The Journal of Immunology 01/2013; · 5.52 Impact Factor
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 190 No. 5 881–892
R. Kang and D. Tang contributed equally to this paper.
Correspondence to Michael T. Lotze: firstname.lastname@example.org; Herbert J. Zeh III:
email@example.com; or Daolin Tang: firstname.lastname@example.org
Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; IP,
immunoprecipitation; MEF, mouse embryonic fibroblast; MEK, MAPK kinase;
mETC, mitochondrial electron transport chain; NAC, N-acetyl cysteine; PARP,
poly(ADP-ribose) polymerase; p-ERK, phosphorylation of ERK; PI, propidium
iodide; ROS, reactive oxygen species; Rot, rotenone; shRNA, short hairpin RNA;
SOD, superoxide dismutase.
During macroautophagy (subsequently referred to simply as
autophagy), subcellular membranes undergo dynamic morpho
logical changes that lead to the engulfment and degrada
tion of cellular proteins and cytoplasmic organelles (Maiuri
et al., 2007; Levine and Kroemer, 2008). Disruption of auto
phagic pathways is associated with multiple disease states,
including neurodegenerative diseases, cancer, infection, and
several types of myopathy (Levine and Kroemer, 2008;
Livesey et al., 2009). Autophagy is also a major mechanism by
which starving cells reallocate nutrients from unnecessary to
more essential processes (Levine and Kroemer, 2008). During
autophagy, a cytosolic form of light chain 3 (LC3; LC3I) is
cleaved and then conjugated to phosphatidylethanolamine to
form the LC3phosphatidylethanolamine conjugate (LC3II),
which is recruited to autophagosomal membranes. Detecting
microtubuleassociated protein LC3 by immunoblotting or
immunofluorescence has become a widely used method for moni
toring autophagy and autophagyrelated processes (Mizushima
and Yoshimori, 2007).
High mobility group box 1 (HMGB1) protein is a highly
conserved nuclear protein, which acts as an architectural
chromatinbinding factor that bends DNA and promotes protein
assembly at specific DNA targets (Lotze and Tracey, 2005).
In addition to its intranuclear role, HMGB1 also functions as
an extracellular signaling molecule during inflammation, cell
differentiation, cell migration, and tumor metastasis (Lotze and
Tracey, 2005; Tang et al., 2010a). HMGB1 is released from
necrotic cells and secreted by activated macrophages, natural
killer cells, and mature dendritic cells, where it mediates the
response to infection, injury, and inflammation (Wang et al.,
1999). In contrast, after DNA damage induced by UV light
irradiation or platination, HMGB1 is sequestered in the nucleus,
which is classically associated with apoptotic, but not necrotic,
cell death (Scaffidi et al., 2002).
ods of starvation and other types of cellular stress. Here
we show that high mobility group box 1 (HMGB1), a
chromatin-associated nuclear protein and extracellular
damage-associated molecular pattern molecule, is a criti-
cal regulator of autophagy. Stimuli that enhance reactive
oxygen species promote cytosolic translocation of HMGB1
and thereby enhance autophagic flux. HMGB1 directly
interacts with the autophagy protein Beclin1 displacing Bcl-2.
utophagy clears long-lived proteins and dysfunc-
tional organelles and generates substrates for
adenosine triphosphate production during peri-
Mutation of cysteine 106 (C106), but not the vicinal C23
and C45, of HMGB1 promotes cytosolic localization and
sustained autophagy. Pharmacological inhibition of HMGB1
cytoplasmic translocation by agents such as ethyl pyru-
vate limits starvation-induced autophagy. Moreover, the
intramolecular disulfide bridge (C23/45) of HMGB1 is
required for binding to Beclin1 and sustaining autophagy.
Thus, endogenous HMGB1 is a critical pro-autophagic
protein that enhances cell survival and limits programmed
apoptotic cell death.
Endogenous HMGB1 regulates autophagy
Daolin Tang,1 Rui Kang,1 Kristen M. Livesey,1 Chun-Wei Cheh,1 Adam Farkas,1 Patricia Loughran,1 George Hoppe,2
Marco E. Bianchi,3 Kevin J. Tracey,4 Herbert J. Zeh III,1 and Michael T. Lotze1
1Damage Associated Molecular Pattern Molecule Laboratory, Department of Surgery, Hillman Cancer Center, University of Pittsburgh Cancer Institute,
University of Pittsburgh, Pittsburgh, PA 15219
2Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195
3Department of Genetics and Cell Biology, San Raffaele University and Research Institute, 20132 Milano, Italy
4Feinstein Institute for Medical Research, Manhasset, NY 11030
© 2010 Tang et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 190 • NUMBER 5 • 2010 882
Figure 1. Autophagy promotes extranuclear HMGB1 translocation and is dependent on ROS generation. (A) HMGB1 translocates from the nucleus to
the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM
rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with
HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by
imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative
images are depicted (right). (B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM
Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific
antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis.
A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with
GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***,
P < 0.0005 versus HBSS group; n = 3. Ctrl, control. (C) Knockdown of ATG5 inhibits LC3-II expression. Western blot analysis of LC3-I/II expression in Panc02
cells under the conditions described in B. Actin was used as a loading control. (D) Hmgb1/ does not influence ATG5 staining. Hmgb1/ and Hmgb1+/+
MEFs were immunostained with HMGB1-specific antibody (green), ATG-5–specific antibody (red), and Hoechst 33342 (dark blue). Representative images
are depicted in the right panels. (E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with
rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the
883 HMGB1 regulates autophagy • Tang et al.
Reactive oxygen species (ROS)–dependent
signals are required for HMGB1
translocation and enhanced autophagy
ROS are signaling molecules important in several pathways that
regulate both cell survival and cell death. Indeed, many stimuli that
induce ROS generation, such as nutrient starvation, mitochondrial
toxins, and hypoxia, also induce autophagy (Fig. S2). ROS forma
tion in the mitochondria is a fundamental regulatory event that pro
motes autophagy (ScherzShouval and Elazar, 2007). The major
source of endogenous ROS is the mitochondrial electron transport
chain (mETC; ScherzShouval and Elazar, 2007).
To evaluate the relationship between mitochondrial ROS
production and HMGB1 translocation during autophagy, we
treated cells with several different mETC inhibitors. Treatment
with rotenone (Rot; complex I inhibitor), thenoyltrifluoroacetone
(complex II inhibitor), and antimycin A (complex III inhibitor)
increased ROS production, LC3 punctae, and LC3II expression
(Fig. 1, E and F). These mETC inhibitors also induced HMGB1
translocation from the nucleus to the cytosol, as assessed by im
aging cytometry and Western blot of subcellular fractions (Fig. 1,
E and F). Under physiological conditions, ROS are cleared from
cells by the action of the superoxide dismutase (SOD) family
members, catalase, or glutathione peroxidase (ScherzShouval
and Elazar, 2007). As expected, knockdown of SOD1 and SOD2
increased starvation and rapamycininduced autophagy and
HMGB1 translocation in Panc2.03 cells (Fig. 1, G and H; and
Fig. S3). Furthermore, Nacetyl cysteine (NAC), an ROS quencher,
dosedependently inhibited starvation and rapamycininduced
autophagy and HMGB1 translocation (Fig. 1, G and H; and
Fig. S3). Moreover, starvation and rapamycin increased ROS/
mitochondrial superoxide levels and decreased SOD activity but
did not affect catalase activity or detected levels (Fig. S2). To
gether, these results are consistent with the notion that ROS signals
are required for HMGB1 translocation and sustained autophagy.
Lack of HMGB1 limits autophagy
To assess the role of HMGB1 in autophagy, we first evaluated
autophagic flux in wildtype and Hmgb1/ MEFs. Hmgb1/
MEFs had markedly diminished LC3GFP punctae, endogenous
LC3 punctae formation, and LC3II expression in cells after
treatment with several autophagic stimuli, including H2O2, rapa
mycin, and starvation (Fig. 2, A–C). Treatment with the lyso
somal protease inhibitors pepstatin or E64D (Mizushima and
Yoshimori, 2007) induced a further increase in LC3GFP punc
tae and LC3II expression in wildtype, but not Hmgb1/,
MEFs (Fig. 2 C). Similarly, knockdown of HMGB1 in HCT116
and Panc2.03 cells also decreased accumulation of LC3 punctae
Although substantial information exists about HMGB1
in the setting of apoptosis and necrosis, the role of HMGB1 in
autophagy is essentially uncharacterized. Here we demonstrate
that HMGB1 is a critical regulator of autophagy, as HMGB1
translocation induces autophagy after prolonged cellular stress.
Interestingly, HMGB1 translocation requires a redoxdependent
signal. Moreover, targeted ablation of HMGB1 increases apop
tosis and inhibits autophagy by sustaining the interaction be
tween Beclin1 and Bcl2. Mutation of cysteine 106 (C106), but
not the vicinal C23 and C45, of HMGB1 promotes cytosolic
localization and autophagy. Additionally, the intramolecular di
sulfide bridge (C23/45) of HMGB1 is required for binding to
Beclin1 and induction of autophagy. These findings define a new
biological function for HMGB1 in promoting cell survival by
sustaining autophagy in response to cellular stress.
Autophagic stimuli promote translocation
of HMGB1 to the cytosol
To investigate the role of HMGB1 when cells are exposed to
stimuli that promote autophagy, we first analyzed its expres
sion and location. Classical autophagic stimuli, such as star
vation (HBSS) or rapamycin treatment, promoted HMGB1
translocation from the nucleus to the cytosol in cultured mouse
embryonic fibroblasts (MEFs), human Panc2.03 (Fig. 1 A),
and other cell lines, such as mouse Panc02, human HCT116,
and mouse RAW264.7 (not depicted). Treatment with rapa
mycin or starvation induced HMGB1 translocation unaccom
panied by measurable lactate dehydrogenase release (Fig. S1),
a marker of plasma membrane disruption. This suggests that
the HMGB1 translocation, observed during early events with
heightened autophagy, is an active process. Furthermore, pre
treatment of the MEF and Panc02 cell lines with the nominal
broad phosphatidylinositol3 kinase inhibitors, such as wort
mannin and Ly294002, blocked starvationinduced accumula
tion of LC3 punctae, LC3II, and HMGB1 translocation (Fig. 1,
B and C). Moreover, knockdown of ATG5, a gene product re
quired for the formation of autophagosomes (Levine and
Kroemer, 2008), significantly inhibited the number of starvation
induced LC3 punctae, LC3II expression, and HMGB1 trans
location (Fig. 1, B and C). This suggests that autophagic stimuli
regulate HMGB1 cytoplasmic translocation. In contrast, there
were no significant differences observed in ATG5 staining
when comparing Hmgb1/ and Hmgb1+/+ immortalized MEFs
(Fig. 1 D), consistent with the notion that ATG5 is not down
stream of HMGB1.
fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific
antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis.
*, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3. (F) mETC inhibitors increase LC3-II expression and promote HMGB1
translocation. Western blot analysis of LC3-I/II and nuclear/cytosolic HMGB1 expression as indicated in E. Fibrillarin is a nuclear fraction control, and
tubulin is a cytoplasmic fraction control. (G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells
were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved
(HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05;
**, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.
(H) Antioxidant and SOD RNAi limit starvation-induced autophagy as measured by LC3-II expression. Western blot analysis of LC3-I/II expression under
the conditions indicated in G. Actin was used as a loading control. Data are means ± SEM.
JCB • VOLUME 190 • NUMBER 5 • 2010 884
Figure 2. Depletion of HMGB1 inhibits autophagy. (A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1/ and HMGB1+/+ MEFs were treated
with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods.
*, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1/
and HMGB1+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A.
(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at
10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units. (D) Up-
regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1/ MEFs were transfected with HMGB1 plasmid or empty vector
885HMGB1 regulates autophagy • Tang et al.
MEFs (Fig. 2 F). These results indicate that HMGB1 is indeed
an important factor that regulates autophagy.
Under normal conditions, 5–10% of HMGB1 protein is
located in the cytosol, with the specific quantity dependent on
the cell type (Fig. 1 A and Fig. 3 A). Moreover, autophagic stim
uli promote translocation of HMGB1 to the cytosol (Fig. 1 A
and Fig. 3 A). To assess the role of cytoplasmic HMGB1 in the
setting of autophagy, we created cytoplasts (anucleate cells).
In response to starvation (HBSS), Hmgb1+/+ MEF cytoplasts
were still able to accumulate LC3 punctae (Fig. 3, A and B;
and expression of LC3II (Fig. S4). Moreover, upregulation of
HMGB1 expression in Hmgb1/ MEFs after gene transfection
restored LC3 punctae formation (Fig. 2 D). Inhibition of autoph
agy leads to an increase in the size and number of p62 bodies
and p62 protein levels (Bjørkøy et al., 2005). Loss of HMGB1
increases the size and number of p62 bodies and p62 protein lev
els (Fig. 2 E), indicating that its degradation is dependent on
HMGB1mediated autophagy. Ultrastructural analysis revealed
that Hmgb1/ MEFs exhibited fewer type I autophagosomes
and type II autophagolysosomes when compared with wildtype
and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3.
Non, nontransfected. (E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml
and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are
depicted (left). (F) Ultrastructural features in Hmgb1/ and Hmgb1+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a–e point to autophago-
somes and autolysosomes). Data are means ± SEM.
Figure 3. Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1/ and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treat-
ment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope.
(A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1/ and Hmgb1+/+ MEFs are depicted. (B) The
percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.
JCB • VOLUME 190 • NUMBER 5 • 2010 886
Bcl2 and Beclin1. The MAPK kinase (MEK)/extracellular sig
nalregulated kinase (ERK) inhibitors PD98059 and U0126 in
hibited starvationinduced phosphorylation of ERK (pERK1/2)
and Bcl2 (Fig. 4 A). Moreover, knockout of HMGB1, as well
as addition of the MEK/ERK inhibitor U0126, blocked the dis
sociation of Bcl2–Beclin1 in the setting of enhanced autophagy
(Fig. 4, B and F), suggesting that ERK1/2mediated phosphory
lation of Bcl2 regulates starvationinduced autophagy. When
present, endogenous HMGB1 demonstrated an interaction with
Beclin1 by coimmunoprecipitation (IP [coIP]) assay in MEFs,
HCT116, and Panc02 cells (Fig. 4, B, C, and E). Knockdown of
Beclin1 inhibited the interaction between Bcl2 and HMGB1 in
untreated HMGB1 wildtype MEFs (Fig. 4, B and D), suggest
ing that HMGB1 interaction with Bcl2 is directly dependent on
Beclin1. Thus, via binding to Beclin1 and regulating the phos
phorylation of Bcl2 in cells after starvation, endogenous
HMGB1 inhibits the interaction between Beclin1 and Bcl2.
Oxidation of HMGB1 regulates
Oxidation of the three cysteines found within HMGB1 is im
portant to its function (Hoppe et al., 2006; Kazama et al., 2008).
Tasdemir et al., 2008). In contrast, Hmgb1/ MEF cytoplasts
demonstrated a lower level of LC3 punctae than HMGB1+/+
(Fig. 3, A and B), indicating that cytoplasmic HMGB1 was re
quired for starvationstimulated autophagy.
Both starvation and rapamycin treatment enhance autoph
agy and induce apoptosis in some cell lines (Maiuri et al., 2007).
We therefore evaluated the relationship between HMGB1 and
apoptosis under these conditions. Increased apoptosis was ob
served in Hmgb1/ MEFs when compared with wildtype
cells by flow cytometry using an annexin V stain, by finding
increased cleaved poly(ADPribose) polymerase (PARP), and
by increased caspase3 activation (Fig. S5). Collectively, these
data suggest that HMGB1 plays a major role in promoting
autophagy and limiting apoptosis.
Depletion of HMGB1 promotes persistent
An important molecular event observed during autophagy is the
disassociation of the Bcl2–Beclin1 complex (Pattingre et al.,
2005). The Bcl2 family of antiapoptotic proteins regulates
Beclin1dependent autophagy (Pattingre et al., 2005). Thus, we
determined the effect of HMGB1 on the interaction between
Figure 4. Absence of HMGB1 sustains Beclin1–Bcl-2 interactions. (A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1/ and HMGB1+/+
MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP
or Western blotting. (B) Knockout of HMGB1 limits the disassociation of the Bcl-2–Beclin1 complex during treatment with autophagic stimuli. HMGB1/ and
HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or
Western blotting. (C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for
protein expression levels as indicated by co-IP or Western blotting. (D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1
and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl,
control. (E and F) Quantitative data demonstrating the interaction between HMGB1–Beclin1 and Beclin1–HMGB1 using densitometry software to assay the
relative protein band intensity in co-IP experiments as shown in A–D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.
887HMGB1 regulates autophagy • Tang et al.
cytoplasmic translocation by ethyl pyruvate (Ulloa et al., 2002)
blocked starvationinduced aggregation of LC3 punctae (Fig. 5 B),
suggesting that cytoplasmic HMGB1 regulates autophagy. Con
sequently, transfection of wildtype HMGB1 and the C106S
variant, but not the mutant C23S or C45S plasmids that are lo
calized to the nucleus, upregulated autophagy and downregulated
apoptosis in Hmgb1/ cells after starvation (Fig. 5, B and C).
The HMGB1 C106S mutation increased the dissociation of
Bcl2 and Beclin1 and promoted pERK1/2 and Bcl2 in
HMGB1/ cells after starvation (Fig. 5 D). The C23S and
C45S variants, but not those with the C106S mutation, impaired
the interaction between HMGB1 and Beclin1 and the pERK1/2
and Bcl2 (Fig. 5 D), suggesting that the intramolecular di
sulfide bridge (C23/45) is required for binding to Beclin1. To fur
ther confirm this hypothesis, we applied a reducing reagent
(DTT) during the IP experiments. As expected, samples treated
with DTT (Fig. 5 E, +DTT) before IP disrupted the interaction
These cysteines are encoded at positions 23, 45, and 106 (C23,
C45, and C106, respectively; Fig. 5 A). Under mild oxidative
conditions, the vicinal cysteines C23 and C45 readily form an
intramolecular disulfide bridge, whereas C106 remains in a re
duced form (Hoppe et al., 2006). Blocking sites of oxidation in
HMGB1 prevents the induction of tolerance by apoptotic cells
(Kazama et al., 2008). Transfection of the constitutively active
redox sensor HMGB1GFPC106 mutation (C106S) into HMGB1
knockout cells impaired nuclear localization of HMGB1, which
increases cytoplasmic aggregation of HMGB1 (Fig. 5 A; Hoppe
et al., 2006) and LC3 punctae formation (Fig. 5 B). This sug
gests that the C106 of HMGB1 may play a role in regulating its
intracellular translocation and, subsequently, autophagy. More
over, autophagic stimuli (rapamycin or starvation) promoted
translocation of wild type as well as mutant C23S, C45S, and
C106S HMGB1 into the cytosol (Fig. 5 A). Inhibition of HMGB1
Figure 5. Oxidation of HMGB1 regulates HMGB1 subcellular localization and autophagy. (A) The C106 mutation (C106S) of HMGB1 impairs its nuclear
localization. Hmgb1/ MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM
rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric
analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The
right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine
mutation locations identified. (B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1/ MEFs were transfected with wild-type
or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were
pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B),
and apoptosis was assayed by FACS (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfec-
tion. (D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1/ MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP
plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or
Western blotting. Blots are representative of three independent experiments with similar results. (E) Reducing reagents disrupt the interaction between wild-
type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as
indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results. (F) Knockdown of Beclin1 by siRNA inhibits
autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or
thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3.
Ctrl, control. Data are means ± SEM.
JCB • VOLUME 190 • NUMBER 5 • 2010 888
factors. Highly integrated and stereotypic response patterns are
found in many organisms, but the means by which so many di
verse pathways, critical for cellular, tissue, and ultimately organ
ismal survival, are coordinated has yet to be elucidated. Here, we
show that depletion of the evolutionarily ancient and highly con
served HMGB1 protein inhibits autophagy in human and murine
cells. Conversely, inhibition of autophagy limits HMGB1 trans
location, suggesting that HMGB1 is central to the regulation of
autophagy. Moreover, we have shown that this effect is controlled
by ERK1/2 phosphorylation and by interrupting the interaction of
Beclin1 with Bcl2. HMGB1 C106 is important for its transloca
tion between the nucleus and cytoplasm, and oxidized C23/45 is
required for the binding to Beclin1. These findings have implica
tions for the temporal and spatial regulation of HMGB1 within
cells and tissues, the connection between HMGB1 and autoph
agic pathways, and the role of HMGB1 as a critical regulator of
HMGB1 protein is both a nuclear DNA binding factor and
a secreted protein that is critically important for cell death and
survival (Tang et al., 2010b). Its activities are determined by
its intracellular localization and posttranslational modifications
(including acetylation, ADPribosylation, phosphorylation, and
thiol oxidation; Scaffidi et al., 2002; Lotze and Tracey, 2005;
Hoppe et al., 2006; Kazama et al., 2008). HMGB1 is released
during necrosis as an endogenous damageassociated molecular
pattern molecule, or “danger” signal, during cell death (Scaffidi
et al., 2002). Latestage apoptotic cells can indeed release
HMGB1, and oxidation of HMGB1 interferes with its ability
to promote immunity (Kazama et al., 2008). ROS also function
between wildtype/C106S HMGB1 and Beclin1. Furthermore,
siRNA knockdown of Beclin1 inhibited autophagy under con
ditions that induce HMGB1 translocation from the nucleus to
the cytosol, such as H2O2, rapamycin, Rot, thenoyltrifluoro
acetone, and HBSS (Fig. 5 F). Moreover, knockdown of Beclin1
inhibited autophagy induced by treatment with exogenous
HMGB1 and overexpression of HMGB1 with or without starva
tion, suggesting that Beclin1 was required for HMGB1mediated
autophagy (Fig. 6, A–D).
To further confirm that oxidation of HMGB1 regulates
autophagic flux, we analyzed the colocalization of lysosomal
associated membrane protein 2 (LAMP2) and LC3 in the pres
ence or absence of bafilomycin A1, an inhibitor of autophagic
vacuole and lysosome fusion. Bafilomycin A1 decreased LAMP2/
LC3 colocalization after starvationmediated autophagy in wild
type MEFs (Fig. 7, A and B). Moreover, expression of HMGB1
or the C106S mutant, but not the C23S and C45S mutants, re
stored autophagic flux in Hmgb1/ MEFs (Fig. 7, A and B).
Together, these findings demonstrate that oxidation of
HMGB1 promotes its localization to the cytosol and subse
quent induction of autophagy (Fig. 8). Our findings also sug
gest that cytoplasmic translocation of HMGB1 is necessary,
but may not be sufficient, to promote and sustain autophagy.
The response of multicellular organisms to stress and mainte
nance of tissue homeostasis is a common biological problem
in all eukaryotes, dictated by both genetic and environmental
Figure 6. Beclin1 is required for HMGB1-mediated autophagy. (A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced
autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting
(A). LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05). (C and D) Knockdown of Beclin1 in MEFs by shRNA
inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle’s balanced salt solution
for 3 h, and LC3 expression was detected by Western blotting (C). LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3;
*, P < 0.05). Data are means ± SEM.
HMGB1 regulates autophagy • Tang et al.
neutrophils and in ischemic tissues, including hepatocytes
(Tang et al., 2007c; Tsung et al., 2007).
These findings (Fig. 2 and Fig. 3) suggest that HMGB1
is directly involved in the positive regulation and maintenance
of autophagy in stressed cells. Autophagy was first discovered
as a nonselective pathway for the degradation of intracellular
constituents, activated in response to starvation (Maiuri et al.,
2007; Levine and Kroemer, 2008). It is now clear that selec
tive autophagic degradation of specific organelles and proteins
occurs in response to diverse stimuli, varying from survival
promoting removal of pathogens, to degradation of damaged
organelles and proteins, to programmed cell survival or cell death
(ScherzShouval and Elazar, 2007). We found that targeted
as signaling molecules in various pathways regulating both cell
survival and cell death. ROS can induce autophagy through sev
eral distinct mechanisms involving catalase activation of Atg4
and disturbances in the mETC (ScherzShouval et al., 2007).
We have shown here that ROS generated during starvation and
rapamycin treatment serve as signaling molecules that initiate
autophagy. Importantly, these stimuli caused HMGB1 cytosolic
translocation from the nucleus, indicating that the function and
localization of HMGB1 necessary to enhance autophagy are
different from that observed in apoptosis. Moreover, we have
demonstrated that HMGB1 translocation in autophagy is ROS
dependent. Indeed, oxidative stress regulates HMGB1 release
and subsequent inflammation in recruited macrophages and
Figure 7. Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1/ MEFs. (A) HMGB1/ MEFs
were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of
100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific
antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly
selected pool of 10–15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro
Plus 5.1 software. Data are means ± SEM.
JCB • VOLUME 190 • NUMBER 5 • 2010 890
It thus appears that cytoplasmic translocation of HMGB1 is
necessary but may not be sufficient to enhance autophagy.
The antiapoptotic molecule Bcl2 is involved in the regu
lation of cell death and survival pathways during apoptosis and
autophagy. It was previously reported that the phosphoryla
tion of Bcl2, likely by kinases of the JNK or ERK signaling
pathway, is required for its antiapoptotic activity (Subramanian
and Shaha, 2007; Wei et al., 2008). Our findings suggest that
HMGB1 may also be involved in the regulation of Bcl2 phos
phorylation by the ERK/MAPK pathway because ablation of
HMGB1 diminishes starvationinduced phosphorylation of both
ERK1/2 and Bcl2, although the mechanism of this effect is not
clear. A recent study has shown that the HMGB1 receptor, recep
tor for advanced glycation endproducts, directly binds to ERK
through a D domain–like docking site and regulates pERK1/2
(Ishihara et al., 2003). It is possible that cytoplasmic HMGB1
interacts with the cytosolic domain of receptor for advanced
glycation endproducts and mediates concomitant ERK1/2 and
Based on the work reported here and previous findings
implicating the nuclear sequestration of HMGB1 in apoptosis,
its extracellular release in inflammation (Wang et al., 1999;
Scaffidi et al., 2002; Kazama et al., 2008), and its critical role as
a universal DNA sensor (Yanai et al., 2009; Sims et al., 2010),
we propose a more nuanced conceptual model involving HMGB1
and cell death. In this model, ROS generated by cellular stress
promote HMGB1 translocation to the cytosol, which induces
autophagy, enhancing ERK signaling and disrupting Beclin1–
Bcl2 complex formation (Fig. 6). Thus, endogenous HMGB1
plays a central role in regulating programmed cell death (apop
tosis) and programmed cell survival (autophagy). These find
ings will be instrumental in developing more effective therapies
in the context of chronic inflammatory diseases, some of them
associated with release of damageassociated molecular pro
teins (Zhang et al., 2010), including neurodegenerative disor
ders, autoimmunity, and cancer.
Materials and methods
The antibodies to HMGB1 were obtained from Sigma-Aldrich and Novus Bio-
logicals. The antibodies to caspase-3, cleaved caspase-3, cleaved PARP, GFP,
Bcl-2, p-ERK (Thr202/Tyr204), and ERK were obtained from Cell Signaling
Technology. The antibodies to tubulin and actin were obtained from Sigma-
Aldrich. The antibodies to phosphoserine, SOD1, SOD2, fibrillarin, and cata-
lase were obtained from Abcam. The antibodies to ATG5 and Beclin1 were
purchased from Novus Biologicals. The antibody to LC3 was purchased from
Novus Biologicals or AnaSpec. The antibody to p62 was obtained from Santa
Cruz Biotechnology, Inc. The SOD activity assay kit was also obtained from
Abcam. The nuclear and cytoplasmic extraction kit was obtained from Thermo
Fisher Scientific. Other reagents and kits were obtained from Sigma-Aldrich.
Panc2.03, Panc02, HCT116, and Hmgb1/ and Hmgb1+/+ immortal-
ized MEF (Scaffidi et al., 2002) cells were cultured in RPMI 1640, DME,
or McCoy’s 5a medium supplemented with 10% heat-inactivated FBS,
2 mM glutamine, and antibiotic–antifungal mix (10,000 units/ml penicillin
and 10,000 µg/ml streptomycin; Invitrogen) in a humidified incubator
with 5% CO2.
Gene transfection and RNAi
HMGB1-GFP or mutant (C23S, C45S, or C106S; Hoppe et al., 2006) or
pUNO1-HMGB1 (InvivoGen) expression vectors were transfected into cells
deletion of HMGB1 limits starvationinduced autophagy and
enhances apoptosis. In the setting of cancer, overexpression of
HMGB1 is associated with aberrant survival of many types of
cancer, including breast, colon, melanoma, and others (Tang
et al., 2010b). Thus, HMGB1 plays an important role in the
regulation of autophagy in response to metabolic stress, oxi
dative injury, and genomic instability promoting programmed
This study demonstrates that HMGB1 promotes and sus
tains autophagy, and we explored the mechanism by which this
occurs. HMGB1 confers proautophagic activities, likely by
controlling Beclin1–Bcl2 complex formation. The dissociation
of Bcl2 from Beclin1 is an important mechanism involved in
activating autophagy and limiting apoptosis in response to star
vation and potentially after other physiological stimuli (Pattingre
et al., 2005). Before this study, the regulation of the interaction
between Bcl2 and Beclin1 by adverse nutrient conditions was
not thoroughly explored. We found that HMGB1 disrupts the
interaction between Beclin1 and Bcl2 by competitively binding
to Beclin1 after starvation. The oxidationsensitive C106, but not
the vicinal C23 and C45, of HMGB1 regulated cytosolic local
ization and autophagy. C106S mutants have much higher cyto
plasmic levels of HMGB1 and demonstrate enhanced binding
to Beclin1, leading to the subsequent dissociation of Bcl2 from
Beclin1. C106S mutants were capable of initiating starvation
induced autophagy as efficiently as wildtype HMGB1, whereas
C23S and C45S mutations abolished this effect. Thus, in two
independent experiments, C106S mutants behave in a similar
fashion to wildtype HMGB1. Meanwhile, C23S and C45S
mutants lose their ability to mediate autophagy, as they are un
able to bind Beclin1 and, therefore, cannot disrupt Bcl2–Beclin1
interactions. These findings demonstrate that oxidation of HMGB1
regulates its localization and ability to sustain autophagy (Fig. 6).
Figure 8. Conceptual relationships between endogenous HMGB1 and
autophagy. ROS trigger HMGB1 translocation to the cytosol in the setting
of starvation-mediated autophagy. Cytosolic HMGB1 then binds Beclin1,
which requires C23/45. This results in dissociation of Beclin1–Bcl-2 and
subsequent induction of autophagy. C106 mutation (C106S) in HMGB1 im-
pairs its nuclear localization and promotes autophagy. Inhibition of HMGB1
translocation by ethyl pyruvate (EP) blocks autophagy. Additionally, HMGB1
promotes phosphorylation and activation of the ERK1/2 (p-ERK1/2) path-
way, which is an important autophagy-dependent signal pathway.
891HMGB1 regulates autophagy • Tang et al.
Transmission electron microscopy assessment of autophagosome and
autophagolysosomes was performed as previously described (Shao et al.,
2004). In brief, cells were fixed with 2% paraformaldehyde and 2% glutar-
aldehyde in 0.1 mol/L phosphate buffer, pH 7.4, followed by postfixation for
6 h in 1% OsO4. After dehydration with graded alcohols, the samples were
embedded in epoxy resin (epon). Then, thin sections (70 nm) were cut with a
microtome (Ultracut R; Leica), mounted on copper grids and poststained with
2% uranyl acetate and 1% lead citrate, dried, and analyzed using a transmis-
sion electron microscope at 25°C (100CX; JEOL, Inc.). Thick sections were cut
(300 nm) and stained with 1% toluidine blue. Images were acquired digitally
from a randomly selected pool of 10–15 fields under each condition.
Apoptosis in cells was assessed using the FITC Annexin V Apoptosis
Detection kit (BD; annexin V–FITC, propidium iodide (PI) solution, and
annexin V binding buffer). This assay involves staining cells with annexin
V–FITC (a phospholipid-binding protein that binds to disrupted cell mem-
branes) in combination with PI (a vital dye that binds to DNA penetrat-
ing into apoptotic cells). Flow cytometric analysis (FACS) was performed
to determine the percentage of cells that were undergoing apoptosis
(annexin V+/PI). Cleaved PARP and cleaved caspase-3 were measured
by Western blotting analysis.
Enucleation by centrifugation
HMGB1/ and HMGB1+/+ MEFs grown on collagen-coated minicover-
slips were enucleated as described previously, with minor modifications
(Goldman et al., 1973; Miller and Ruddle, 1974). To enucleate the cells,
the minicoverslips were inverted (cell side down) and placed into the bot-
tom of 1.5-ml Eppendorf tubes containing 10 µg/ml cytochalasin B (Sigma-
Aldrich) in medium. After centrifugation at 12,000 g for 60 min, the
minicoverslips were removed from the tubes and placed cell side up into
a 6-well plate containing 2 ml of medium without cytochalasin B for sub-
sequent studies and staining for autophagy.
Data are expressed as means ± SEM of three independent experiments
performed in triplicate. One-way analysis of variance was used for com-
parison among the different groups by SPSS 12.0. When the analysis of
variance was significant, post-hoc testing of differences between groups
was performed using Fisher’s least significant difference test. A p-value
<0.05 was considered significant; P < 0.005 and P < 0.0005 were re-
ported where applicable.
Online supplemental material
Fig. S1 shows that autophagic stimuli promote cytosolic HMGB1 transloca-
tion that is not dependent on plasma membrane disruption. Fig. S2 shows
that autophagic stimuli increase ROS and mitochondrial superoxide levels
and decrease SOD activity while having no direct effect on catalase activ-
ity levels. Fig. S3 shows that provision of antioxidants or knockdown of
SOD1 and SOD2 increases rapamycin-induced autophagy and HMGB1
translocation. Fig. S4 shows that knockdown of HMGB1 limits autophagy
in HCT116 and Panc2.03 cells. Fig. S5 shows that HMGB1 depletion pro-
motes apoptosis. Online supplemental material is available at http://www
Thoughtful discussions and review of this work with Timothy Billiar and Sarah
Berman at the University of Pittsburgh and external colleagues Guido Kroemer,
Douglas Green, Matthew Albert, Eva Szegezdi, and Beth Levine were much
appreciated. We also thank the reviewers for constructive suggestions.
This project was funded by the National Institutes of Health (an Integrat-
ing NK and DC into Cancer Therapy grant [1 P01 CA 101944-04] from the
National Cancer Institute to M.T. Lotze) and a grant from Associazione Italiana
Ricerca sul Cancro (to M.E. Bianchi).
Submitted: 16 November 2009
Accepted: 3 August 2010
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incubated for 2 h at 25°C or overnight at 4°C with various primary anti-
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Measurement of intracellular ROS and mitochondrial superoxide
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stimuli for the indicated time. ROS were assessed with cell-permeable dye
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(Invitrogen) as described previously (Kang et al., 2010b). Analysis of sig-
nal intensity was performed using a fluorescent plate reader.
Cells were lysed at 4°C in ice-cold radioimmunoprecipitation assay lysis buffer
(Millipore), and cell lysates were cleared by a brief centrifugation for 10 min
at 12,000 g. Concentrations of proteins in the supernatant were determined
by bicinchoninic acid assay. Before IP, samples containing equal amounts of
proteins were precleared with protein A or protein G agarose/Sepharose at
4°C for 3 h and subsequently incubated with 2–5 µg/ml various irrelevant IgG
or specific antibodies in the presence of protein A or G agarose/Sepharose
beads for 2 h or overnight at 4°C with gentle shaking (Tang et al., 2007a,b).
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Cells were seeded in 96-well plates, cultured in the presence of various stimuli
for given times, and then were fixed with 3% paraformaldehyde and stained
with HMGB1 or LC3 antibody. Secondary antibodies were goat IgG conju-
gated either with Alexa Fluor 488 or Alexa Fluor 647 fluorochromes. Nuclear
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tial distribution of fluorescently labeled components in cells placed in 96-well
microtiter plates. The Spot Detector BioApplication (Thermo Fisher Scientific)
was used to acquire and analyze the images after optimization. Images of
500–1,000 cells for each treatment group were analyzed to obtain the mean
nuclear and cytosolic HMGB1 intensity and LC3 fluorescence punctae number
per cell (Tang et al., 2009).
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cytometry as previously described (Kang et al., 2010a). Formation of
autophagic vesicles was further monitored by transient expression of GFP-
LC3 (gifts of X.-M. Yin, University of Pittsburgh, Pittsburgh, PA) aggregation
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by counting the number of positively staining cells from 100 randomly
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cal microscope (Fluoview FV-1000; Olympus) using a 60x Plan Apo/1.45
oil immersion objective at 25°C and were captured and analyzed by
Fluoview software (FV10-ASW 1.6; Olympus). Images were subsequently
analyzed for the level and colocalization by Image-Pro Plus 5.1 software
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