Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress
et al. (2013) Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic
Stress. PLoS ONE 8(4): e62758. doi:10.1371/journal.pone.0062758
Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress
Kurosh Ameri 0
Anthony M. Rajah 0
Vien Nguyen 0
Timothy A. Sanders 0
Arman Jahangiri 0
Michael DeLay 0
Matthew Donne 0
Hwa J. Choi 0
Kathryn V. Tormos 0
Yerem Yeghiazarians 0
Stefanie S. Jeffrey 0
Paolo F. Rinaudo 0
David H. Rowitch 0
Manish Aghi 0
Emin Maltepe 0
Rajesh Mohanraj, UAE University, United Arab Emirates
0 1 Department of Pediatrics/Neonatology, University of California San Francisco , San Francisco , California, United States of America, 2 Department of Neurological Surgery, University of California San Francisco , San Francisco , California, United States of America, 3 Department of Developmental and Stem Cell Biology, University of California San Francisco , San Francisco , California, United States of America, 4 Department of Medicine/Cardiology, University of California San Francisco , San Francisco , California, United States of America, 5 Department of Surgery, Stanford University School of Medicine, Palo Alto, California, United States of America, 6 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco , San Francisco, California , United States of America
Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosisinducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1a (HIF1a). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.
Funding: Funding provided by Department of Pediatrics, UCSF and by NIH (HL087754, HD072455, T32HD007470). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Ischemic heart disease, stroke and cancer are associated with
cellular hypoxia and nutrient/glucose deprivation [1,2,3,4]. The
Hypoxia Inducible Factor (HIF) family of transcriptional
regulators modulates the survival of cells in response to these stressors 
[6,7,8]. HIFs are heterodimers consisting of oxygen sensitive,
labile a subunits complexed with stable b subunits. With
increasing levels of oxygen, HIF-a subunits are hydroxylated at
conserved proline residues, mediated by a family of
prolyl-4hydroxylase domain (PHD) enzymes. Hydroxylated HIFa is then
recognized and targeted for proteasomal degradation by the von
Hippel-Lindau protein (pVHL) complex. Under hypoxic
conditions, PHD activity ceases and the rate of hydroxylation declines
leading to HIF-a accumulation [9,10,11]. Once stabilized, HIF-1a
heterodimerizes with HIF-1b, and regulates the expression of
scores of adaptive/survival genes. Therapeutic manipulation of
HIF-hydroxylases therefore has obvious appeal .
The maintenance of cellular bioenergetics within tissues and
organs is dependent on the coordinated interplay between multiple
competing factors. Variations in substrate delivery and cellular
metabolic rates can produce wide ranges of tissue oxygenation
even in adults during non-stressful steady states [13,14].
Furthermore, all of mammalian development occurs in a physiological
hypoxia that does not compromise normal growth, but that is still
dependent on HIF [15,16]. Thus, cells possess multiple
compensatory mechanisms to preserve cellular bioenergetics across a wide
range of oxygen and glucose concentrations, and hypoxia and/or
glucose deprivation only become pathologic when these
countermeasures are exhausted . During anoxia or ischemia,
conditions that limit mitochondrial ATP production, adaptive
mechanisms fail and cells undergo an adaptation-to-death
switch [2,18,19], frequently in advance of true bioenergetic
collapse. Interestingly, some cancer cells can escape this switch due
to malfunctioning death pathways that contribute to their
malignant progression [20,21,22].
Following bioenergetic compromise, multiple forms of cell death
such as programmed cell death (PCD), or apoptosis, as well as
necrosis are observed. In general, PCD can be classified as
caspase-dependent or independent . Subcellular
relocalization of effector proteins frequently drives these processes. In the
intrinsic form of PCD, for example, signals from mitochondria,
such as cytochrome c, are liberated to induce downstream caspase
activation and subsequent cell death . The export of
cytochrome c during apoptosis is regulated by mitochondrial
outer membrane permeabilization. This is determined, in part, by
the opposing actions of the BCL-2 family of mitochondrial outer
membrane proteins . For example, while BCL-XL inhibits
mitochondrial outer membrane permeabilization, BAX and BAK
promote it . Similarly in caspase-independent PCD,
mitochondrial factors such as AIF are released in response to toxic
stimuli and directly promote apoptotic cell death following nuclear
translocation . During this adaptation-to-death switch,
therefore, several factors contribute to cell death pathways via
mechanisms dependent on altered subcellular localization
Higd1a is a HIF-1 target gene originally described in cultured
human cervical epithelial cells , and shown to be induced in
hypoxic neuron-enriched primary cultures  as well as by nickel
in mouse embryo fibroblasts . HIGD1A is a ,10 kDa
mitochondrial inner membrane protein with adaptive functions
during glucose deprivation , and promotes normal
mitochondrial function via modulation of the mitochondrial c-secretase
complex . The survival effect of HIGD1A is dependent on the
level of HIGD1A expression . Anti-apoptotic effects of
HIGD1A in RAW264.7 macrophages have been shown to be
associated with inhibition of cytochrome C release and reduced
caspase activation . In the rat spinal cord, HIGD1A
expression increases after birth and during the first days of
postnatal life during CNS remodeling . During this period,
many populations of neurons are known to undergo cell death
with the number of apoptotic cells peaking just after birth and
falling sharply the week thereafter. This trend suggests both cell
death and survival roles for HIGD1A, depending on
developmental stage and cellular microenvironment.
The subcellular localization of HIGD1A during severe stress has
not been addressed to date. In this paper, we have investigated the
localization of HIGD1A in mouse embryonic fibroblasts (MEFs)
during metabolic stress, including glucose starvation coupled with
prolonged hypoxia, in addition to etoposide induced DNA
damage. We also examined the subcellular localization of
HIGD1A during pathological states in vivo, including in human
neonatal brains following HIE and infarcted mouse hearts, as well
as human tumor xenografts and glioblastoma biopsies from
patients before and after treatment with the antiangiogenesis
agent Bevacizumab/Avastin. While found in mitochondria under
basal conditions, we found that HIGD1A was frequently localized
to the nucleus during these metabolically stressful states.
Interestingly, HIGD1A and AIF interacted, and their nuclear localization
was dependent on BAX and BAK. In summary, we describe a
novel subcelluar localization for HIGD1A in the nucleus during
severe stress in vitro and in several pathologic conditions associated
with severe hypoxia and ischemia in vivo.
HIGD1A is Regulated by HIF1a and Localizes to the
Nucleus during Severe Stress
To determine whether HIGD1A expression and induction is
regulated by HIF1a or HIF2a, we used HIF-deficient MEFs and
trophoblast stem cells (TSCs). As indicated by RTPCR in Fig. 1A,
in contrast to wt cells (HIF+/+), HIF-1a deficient MEFs (HIF2/
2) failed to induce Higd1a mRNA in hypoxia (Fig. 1B). Similarly,
HIGD1A protein was only induced in wt (+/+) cells (MEFs and
TSCs) when subjected to hypoxia (1% O2), but not in HIF
deficient MEFs (2/2) or HIF-1/2a deficient TSCs (2/2). To
determine whether HIGD1A was regulated specifically by HIF1a
or HIF2a, we overexpressed HA-tagged HIF1a and HIF2a in
HIF deficient TSCs (Hif-1/2a2/2) as previously described .
GFP overexpression in the same plasmid backbone served as a
control. As indicated in Fig. 1C, HIGD1A protein was induced
only when HIF1a was overexpressed. This induction of HIGD1A
by HIF1a was dependent on canonical hypoxia response element
binding, since overexpression of HIF1a that lacked the DNA
binding basic domain (HIF-1aDb) failed to induce HIGD1A.
These results demonstrate that HIGD1A is exclusively regulated
by HIF1a via canonical target gene expression.
Several factors such as AIF  or GAPDH [28,31,34] become
nuclear when cells are subjected to severe stress, such as during
ischemia or exposure to DNA damaging agents such as etoposide.
As shown in Fig. 1D, during physiological hypoxia (2% O2),
endogenous HIGD1A was primarily localized to mitochondria in
MEFs, confirming previous results. When subjected to ischemia
(1% oxygen coupled with glucose starvation), or, as shown in
Fig. 1E, the DNA damaging agent etoposide, however,
endogenous HIGD1A localized to the nucleus, whereas complex IV
subunit 2 of the electron transport chain remained mitochondrial
under all conditions. To confirm these observations made with
endogenous HIGD1A, and to rule out non-specific staining
artifacts, we also examined MEFs that stably overexpressed a
HIGD1A-GFP fusion protein. As indicated by live-cell
epifluorescence microscopy in Fig. 1F, control cells prior to etoposide
treatment demonstrated mitochondrial/cytoplasmic
HIGD1AGFP fluorescence. However, as early as 2 hours following
treatment with etoposide, nuclear entry of HIGD1A-GFP fusion
protein could be demonstrated, which increased throughout the
duration of the experiment.
HIGD1A Interacts with AIF and its Nuclear Localization is
Dependent on BAX and BAK
To determine whether HIGD1A nuclear localization was
associated with the nuclear translocation of AIF, we treated
HIGD1A-GFP overexpressing cells with etoposide, and
costained for GFP and AIF. As indicated in Fig. 2A, AIF and
HIGD1A co-localized to mitochondria in untreated control
cells. However, upon exposure to etoposide, co-staining for
HIGD1A-GFP and AIF demonstrated the presence of both
factors within the nucleus. Quantitation of nuclear HIGD1A
relative to untreated control cells demonstrated significantly
greater numbers of cells with nuclear HIGD1A when cells were
treated with etoposide. Confocal immunofluorescence
microscopy confirmed the nuclear col-localization of AIF and HIGD1A
in response to Etoposide (Fig. 2B). We confirmed these
observations of nuclear HIGD1A accumulation via biochemical
fractionation followed by immunoblot analyses. As shown in
Fig. 2Ci, in untreated control cells, HIGD1A-GFP fusion
protein was localized primarily within mitochondrial fractions,
although a small amount of cytoplasmic HIGD1A was also
appreciated. Following etoposide treatment, however, a clear
nuclear accumulation of HIGD1A was also appreciated, along
with nuclear GAPDH (Fig. 2C). As subcellular markers, we used
Histone H3 (H3), which is a nuclear protein, electron transport
chain complex IV subunit 2, which is a mitochondrial protein,
and GAPDH, which can localize to both the cytoplasm as well
as mitochondria, and is known to translocate to the nucleus
during severe stress [28,31,34]. As indicated in Fig. 2C, only
HIGD1A and GAPDH became nuclear after etoposide
treatment, whereas Histone H3 was solely present in the nucleus,
and complex IV subunit 2 of the respiratory chain was only
localized to mitochondria, irrespective of etoposide treatment.
Together, these results confirm that HIGD1A is primarily a
mitochondrial factor under basal conditions, but also
accumulates in nuclei when cells experience severe stress.
Since AIF and HIGD1A were frequently observed within the
same subcellular compartments under physiological and
pathological conditions, we questioned whether HIGD1A and AIF
might physically interact. As indicated in Fig. 2C ii, we were
able to identify AIF as a HIGD1A-interacting protein following
immunoprecipitation. Neither BNIP3, another mitochondrial
HIF-1 target, nor VDAC, another mitochondrial outer
membrane protein , interacted with HIGD1A, highlighting the
specificity of the observed HIGD1A-AIF interaction.
Nuclear localization of AIF has been reported to be
dependent on the presence of BAX and BAK . We
therefore interrogated BAX/BAK double knock out MEFs
(Bax/Bak2/2) for nuclear localization of AIF and HIGD1A
during etoposide-induced stress. As indicated in Fig. 2D, when
Bax/Bak2/2 cells were treated with etoposide, nuclear
localization of AIF and HIGD1A was diminished when compared with
wt MEFs. Quantitation of nuclear HIGD1A relative to
untreated control cells demonstrated no significant differences
between Bax/Bak2/2 cells treated with etoposide and control
cells. These results suggest that the nuclear localization of
HIGD1A is dependent on BAX and BAK activity.
HIGD1A Localizes to the Nucleus during Human Neonatal
Hypoxic-ischemic Encephalopathy (HIE) in vivo
To investigate the relevance of nuclear HIGD1A localization
in vivo, we examined tissue samples obtained from pathological
conditions associated with hypoxia/ischemia, including HIE.
Specifically, the sub-venticular zone (SVZ) of the brain was
examined (Fig. 3A). As shown in Fig. 3B, the SVZ of human
neonatal brains obtained from babies that succumbed to HIE were
hypoxic as indicated by greater staining for carbonic anhydrase 9
(CA9)a hypoxia marker regulated by HIF-1 . As indicated in
Fig. 3C, these regions demonstrated nuclear staining of HIGD1A,
whereas control brains demonstrated weaker, non-nuclear
HIGD1A Localizes to the Nucleus during Murine
Myocardial Infarction (MI) in vivo
To investigate the relevance of nuclear HIGD1A in vivo
further, we examined infarcted mouse hearts generated utilizing
a total occlusion model . As an internal control for presence
of ischemia we stained for AIF, which is known to translocate
to the nucleus during severe ischemia . As shown in Fig. 4,
peri-infarct areas demonstrated diffused and nuclear AIF,
whereas sites distal to the infarct demonstrated distinctly
extranuclear AIF localization. Similar to these results,
myocardial tissue surrounding the necrotic core of infarcted hearts
demonstrated robust nuclear HIGD1A staining, whereas
noninfarcted distal regions showed extranuclear HIGD1A
localization. These in vivo results, together with the results in Fig. 3,
suggest that nuclear localization of HIGD1A might be a
widespread phenomenon during severe stress, and could
potentially serve as a biomarker during these conditions.
HIGD1A Localizes to the Nucleus in Peri-necrotic Tumor
Regions in Cancer Xenografts in vivo
Due to their rapid growth rates and defective vascularity, solid
tumors are heterogeneous with respect to tissue oxygen and
nutrient delivery. We examined HIGD1A expression in the
human triple negative invasive breast cancer MDA-MB 231
xenografts that have previously been characterized and shown to
contain anoxic perinecrotic regions . As indicated by H&E
staining in Fig. 5A, tumors contained necrotic regions.
Perinecrotic regions, which are known to be severely hypoxic, stained
Figure 3. HIGD1A localizes to the nucleus in the setting of human neonatal hypoxic-ischemic encephalopathy (HIE) in vivo. (A)
Schematic depiction of a coronal section through a human neonatal brain highlighting the subventricular zone (SVZ). (B) The SVZ of brains obtained
from infants with HIE exhibited increased levels of the hypoxia marker CA9 compared with non-HIE control brains. (C)Immunofluorescence
microscopy indicated low-level, extra-nuclear localization of endogenous HIGD1A in control human neonatal brains. Endogenous HIGD1A levels are
increased in regions of human neonatal brains of infants who suffered HIE. Arrows indicate nuclear localization of endogenous HIGD1A in each.
Experimental observations were made at least three times, and in vivo patient data are representative of three cases.
positive for the endogenous hypoxia marker CA9 as indicated by
immunofluorescent microscopy. These same areas also stained
strongly for HIGD1A. Regions distal to tumor necrotic areas
stained weakly for CA9 and HIGD1A. As shown in Fig. 5B,
perinecrotic tumor areas demonstrated nuclear HIGD1A
localization, whereas distal regions to necrotic cores contained
predominantly extranuclear HIGD1A.
HIGD1A Localizes to the Nucleus in Human
Glioblastomas after Antiangiogenesis Treatment
Anti-angiogenesis is currently being used in cancer therapy to
disrupt tumor vascularization, which can result in cancer cell
death due to induction of anoxia and severe ischemia. To assess
the relevance of nuclear HIGD1A location in antiangiogenesis
therapy, we first examined HIGD1A expression in glioblastoma
xenografts before and after administration of Bevacizumab
(Avastin). As indicated in Fig. 6A, before administration of
Bevacizumab, HIGD1A was primarily extranuclear. However,
after Bevacizumab treatment, HIGD1A also localized to the
nucleus in these xenografts.
We further investigated the in vivo relevance of our xenograft
observations in a human therapeutic setting that triggers
significant tumor anoxia, ischemia, and hence, glucose starvation.
Adaptive mechanisms that allow tumor cell survival following
antiangiogenesis treatments can compromise their therapeutic
efficacy, highlighting the importance of understanding these survival
pathways [52,53] . Therefore, to determine if HIGD1A was
similarly induced in human glioblastomas following
anti-angiogenesis treatment in vivo, we examined HIGD1A expression in
human glioblastoma biopsies obtained before and after
administration of Bevacizumab (Avastin) to patients. As shown in Fig. 6B,
prior to the administration of Bevacizumab, both the hypoxia
marker CA9 and HIGD1A levels were low. Localization of
HIGD1A was primarily non-nuclear. However, after
administration of Bevacizumab, hypoxic areas where created as indicted by
increased CA9 staining. Under these conditions, HIGD1A
expression was significantly increased, and was localized primarily
to the nucleus, correlating with severe metabolic stress.
Figure 4. HIGD1A localizes to the nucleus in the setting of murine myocardial infarction (MI) in vivo. Top panel is a representative H&E
stain of a mouse heart subjected to MI highlighting the area of infarct as well as regions distal to it where tissue was analyzed. As seen, in areas distal
to the infarct, HIGD1A and AIF are expressed in a primarily extranuclear distribution. In the area of infarct, however, HIGD1A and AIF exhibit a much
more diffuse localization that clearly includes nuclei (arrows).
In this study, we have demonstrated the stress dependent
nuclear localization of the HIF-1 target mitochondrial protein
HIGD1A in vitro and in vivo. While physiological hypoxia promotes
mitochondrial HIGD1A expression in a HIF-1-dependent
manner, we found that severe metabolic stressors such as ischemia or
DNA-damaging agents such as etoposide trigger nuclear
accumulation of HIGD1A. Several mitochondrial factors such as AIF 
and GAPDH  also become nuclear during conditions of severe
stress, and the nuclear function of these factors modulates cell
death pathways. While HIF-1 is generally considered to be an
adaptive factor promoting cell survival during hypoxia, it can also
promote cell death pathways via its target genes. BNIP3 is a
mitochondrial factor [56,57], and its expression is regulated by
HIF-1 . BNIP3 has primarily been described as a death factor,
promoting apoptosis or autophagy [59,60], although protective
roles have also been described, depending on its subcellular
localization. For example, in glioblastomas, BNIP3 has recently
been localized to the nucleus , where it binds to the Aif gene
promoter and represses its expression, thereby inhibiting
AIFmediated cell death [29,30]. Further complicating the picture, AIF
appears to also have dual nuclear roles. AIF can translocate from
mitochondria to the nucleus and either induce apoptosis  or
autophagy , which can promote cell death or survival,
Figure 5. Nuclear localization of HIGD1A in mouse models of human breast cancer xenografts. (A) Top panel is a representative H&E
stained slide of a human breast cancer xonograft indicating the perinecrotic region surrounding the necrotic core, as well as areas distal to the
necrosis. Immunofluorescence microscopy analysis indicated that HIGD1A and the hypoxia marker CA9 were only minimally expressed distal to the
region of necrosis, whereas both were highly expressed in the peri-necrotic region. (B) Perinecrotic regions contained predominantly nuclear (white
arrows) localized HIGD1A, whereas areas distal to tumor necrotic regions had predominantly extranuclear HIGD1A. (B) Immunofluorescence
microscopy of human gliobastoma xenografts demonstrating predominantly extranuclear HIGD1A before administration of Bevacizumab
(preBevacizumab), whereas after administration of Bevacizumab (post-Bevacizumab), HIGD1A becomes predominantly nuclear as indicated by white
respectively. Similar to AIF and BNIP3, GAPDH is a mobile
factor within the cell. Nuclear GAPDH can participate in cell
death/dysfunction [31,34], but can also have roles in cell survival
via activation of DNA repair mechanisms, maintenance and
protection of telomeric DNA from rapid degradation, and
regulation of the redox state of a number of transcriptional
regulators [28,33,62,63]. The Hsp90-binding immunophilin
FKBP51 is another mitochondrial protein that similarly becomes
nuclear during stress, which then protects against oxidative stress
. Like AIF, GAPDH or FKP51, HIGD1A might also have
novel nuclear roles that could fine tune cellular fates during
conditions of severe stress.
Nuclear localization of mitochondrial proteins such as AIF is
regulated in part by BAX and BAK mediated modulation of the
mitochondrial outer membrane permeability [48,65]. Our results
suggest that nuclear localization of HIGD1A is similarly regulated
by the presence of BAX and BAK. While AIF and GAPDH are
believed to translocate directly from mitochondria to the nucleus,
the localization of HIGD1A to the inner mitochondrial membrane
makes this mechanism less likely. We surmise that a separate
cytosolic pool of HIGD1A translocates to the nucleus during
severe stress. Our biochemical fractionation experiments support
this hypothesis, as mitochondrial HIGD1A levels did not decrease
during apoptosis induction in MEFs (Fig. 2Ci).
Similar to our in vitro results, we also demonstrate
nuclearlocalization of HIGD1A during severe stress in vivo. Specifically,
we show that in the setting of ischemic heart disease,
hypoxicischemic encephalopathy and cancer, nuclear localization of
HIGD1A correlates with severity of stress. Other HIF-1 targets
such as Carbonic anhydrase 9 are endogenous markers of hypoxia
and are upregulated in tumors after anti-angiogenesis treatment,
and enable cell survival [52,53]. Whether nuclear HIGD1A also
promotes increased cell survival in these settings remains to be
elucidated. Our results suggest, however, that it may potentially be
a useful biomarker of pathological hypoxic/ischemic states in vivo.
Neonatal brain tissue was collected with written informed
consent in accordance with guidelines established by the
University of California San Francisco Committee on Human
Research (Institutional Review Board IRB# H11170-19113-07).
The CHR reviews research involving human subjects to ensure
the ethical and equitable treatment of those subjects. Human tissue
was obtained from autopsied material at the University of
California San Francisco Medical Center following the general
guidelines posted on http://www.research.ucsf.edu/chr/Guide/
Figure 6. Nuclear localization of HIGD1A in response to Bevacizumab in human glioblastoma xenografts as well as glioblastoma
patient biopsies. (A) Immunofluorescence microscopy of human glioblastoma xenografts showing HIGD1A expression and localization before (pre)
and after (post) Bevacizumab treatment. White arrows indicate nuclear HIGD1A. (B) Immunofluorescence microscopy of paired human patient
gliobastoma biopsies showing CA9 (hypoxia marker) and HIGD1A expression and localization before (pre) and after (post) treatment with the
antiangiogenic agent, Bevacizumab (Avastin). As indicated, HIGD1A was induced and predominantly nuclear in human glioblastoma samples after
administration of Bevacizumab to patients. Lower levels of HIGD1A was expressed before treatment. As indicated in the inset HIGD1A localization to
the nucleus is pronounced in glioblastoma after treatment with Bevacizumab (white arrows).
Information about bevacizumab-resistant cases was obtained as
part of a study approved by the UCSF Committee on Human
Research (CHR). The CHR reviews research involving human
subjects to ensure the ethical and equitable treatment of those
subjects. Tissue from these cases was acquired from the UCSF
Brain Tumor Research Center (BTRC), which obtains tissue after
obtaining written informed consent from patients, a consent which
allows the BTRC to distribute tissue to UCSF investigators.
Human brain tumor tissue was obtained at the University of
California San Francisco Medical Center following the general
guidelines posted on http://www.research.ucsf.edu/chr/Guide/
Cell Culture Conditions and Chemicals
Mouse embryonic fibroblasts (MEFs) were cultured in
RPMI1640 (Lonza), 10%FBS, 2.5 mg/ml Fungizone, 100 mg/ml
Penicillin/Streptomycin, and 110 mg/ml Sodium Pyruvate. MEFs
have been described in . Bax/Bak2/2 MEFs were from
obtained from N. Chandel. Fungizone, Penicillin/Streptomycin,
and Sodium Pyruvate were from the UCSF Cell Culture Facility.
Glucose starvation was achieved by culturing cells in MEF media
utilizing glucose free RPMI 1640 (Lonza). Cells were harvested via
trypsinization using 0.25% trypsin with EDTA also sourced from
the UCSF Cell Culture Facility. Cells were incubated in a tissue
culture incubator at 5% CO2 and 21% O2 while hypoxic
experiments were performed for 20 hours at 2% or 1% O2 with
5% CO2 using a HERA-cell 240 (Thermo Electron Corp), or an
XVivo hypoxia workstation(Biospherix). Oxygen level was
monitored with inbuilt oxygen sensors or by using an Analox oxygen
indicator (Analox). Cells were incubated in RPMI for 24 hours
and 40 mM etoposide (Sigma) was added to the cells for indicated
Cellular Fractionation Extracts
Cells were seeded overnight to achieve a density of
approximately 80%, and then treated with etoposide for 12 hours.
Fractions were made by using the MS861 Cell Fractionation Kit
per manufacturers instructions (MitoSciences), with slight
modification, where the final nuclear pellet was completely lysed with
the use of a sonicator.
For SDS-PAGE, whole cell lysates were prepared in a cold
room (4uC). Lysates were prepared by using Urea lysis buffer (8 M
urea, 10% glycerol, 5 mM DTT, 10 mM Tris-HCl pH 6.8, 1%
SDS, and 1x Proteinase Inhibitor Cocktail (Roche Diagnostics)) by
adding lysis buffer directly to cells that were washed with ice cold
PBS. Lysates were sonicated to ensure complete lysis. Protein
levels were quantified using the bicinchoninic acid (BCA) Protein
Assay (Thermo Scientific Pierce). Whole cell lysates (20100 mg
protein per lane) were subjected to gel electrophoresis on 7.5%,
12% or 15% Gold Pre Cast PAGEr gels (Lonza) and blotted onto
Immobilon-FL membranes (Millipore) using semi-dry transfer
(Bio-Rad). Membranes were blocked in blocking buffer from
LICOR Biosciences and probed with primary antibodies in LI-COR
blocking buffer. Primary antibodies were: murine HIGD1A
(Proteintech Group), human HIGD1A (Santa Cruz), GFP
(Invitrogen), GAPDH (Novus), Complex IV (Mitosciences),
Histone H3 (Abcam), AIF (Cell Signaling), BNIP3 Cell Signaling).
For secondary antibodies, IRDye 800CW goat anti-rabbit and
IRDye 680 goat anti-mouse secondary antibodies (LI-COR
Biosciences) were used in LI-COR blocking buffer supplemented
with SDS and Tween-20 according to the manufacturers
protocol. Proteins were visualized in conjunction with the
LICOR Odyssey Imaging System for signal detection.
The murine HIGD1A cDNA was obtained from Origene
(Image accession number 5148784) and used for all subsequent
expression constructs. HIGD1A fusion proteins were generated
through overlap extension PCR, cloned into the ENTRD-TOPO
vector (Invitrogen) and confirmed by DNA sequencing. For
HIGD1A-GFP, monomeric EGFP (Karel Svoboda, Addgene
Plasmid 18696) was fused to the C-terminus of HIGD1A via a
25 amino acid tetrameric helical linker (HL4) . For expression
in cell culture, a derivative of the Piggybac transposon system from
 was employed allowing high efficiency expression. The
parental plasmid EBXN containing the minimal Piggybac 59 and
39 inverted terminal repeats as well as a CMV enhancer chicken
Beta-actin promoter expression cassette was modified to include
the SV40 promoter Blasticidin cassette allowing for eukaryotic
selection in cell culture. The plasmid was further modified to
include the Invitrogen Gateway Rfa cassette allowing for phiC31
mediated recombination. HIG1DA-GFP was cloned into PBX2.2.
Transfection was performed with Lipofectamine LTX and
PLUS reagent (Invitrogen). A 2:1 molar ratio of Piggybac
transposase helper plasmid was combined with the transposon
expression construct to mediate integration and high level
expression. Selection with Blasticidin 10 mg/ml was performed
to select for stable integrants. Selected cells for expression of GFP
or HIGD1A-GFP were frozen and thawed when needed for
Immunohistochemistry and Microscopy
Staining of cells: Cells grown on microscope cover glass were
fixed in ice cold methanol for 15 minutes at 220uC, after which
they were washed with PBS. Blocking was performed in BSA/
PBS/Tween20 for 1 hour, after which primary antibody (1:300
dilution) in BSA/PBS without Tween20 was added to the cells for
1 hour. Cells were then washed with PBS, and then secondary
AlexaFluor antibody was added in BSA/PBS/Tween20 solution
for 1 hr. Cells were washed with PBS and mounted with
Vectashield mounting solution containing DAPI.
Staining of tissue samples: Neonatal HIE brain cryosections
were cut at 20 mm. Paraffin embedded heart and MDA-MB 231
tumor sections were cut at 2 and 5 mm respectively. Glioblastoma
paraffin embedded sections were cut at 16 mm. Paraffin embedded
sections were heated to 95uC for antigen retrieval in 0.01 M
Citrate buffer, pH 6.0, and blocked with 10% normal goat serum
in 1.5% Triton X-100/PBS for 1 hour at room temperature.
Sections were incubated overnight at 4uC in primary antibody in
10% goat serum and 0.5% Triton X-100/PBS. AlexaFluor
Secondary fluorescent antibodies (Invitrogen) were used for
Cells were visualized in the UCSF Biological Imaging
Development Center utilizing a spinning disk confocal microscope (Zeiss
Axiovert microscope, Yokogawa CSU10 confocal scanner unit), or
with a Zeiss Imager Z.2 fluorescence microscope (Karl Zeiss)
equipped with an Apotome and axiovision software for optical
Patient Glioblastoma. Additional information on
Glioblastoma biopsies and bevacizumab treatments can be obtained in
detail from [52,69]. Briefly, tumors from patients at recurrence but
before bevacizumab treatment were as pre-Avastin/bevacizumab
section. Avastin was administered at 10 mg/kg and tumors were
followed with MRI. Once the tumor became resistant (no longer
shrinking, more infiltrating) tumors were surgically removed and
harvested with the resulting tissue considered post-bevacizumab.
For glioblastoma xenografts, 500 K U87 MG tumor cells were
injected subcutaneously into 10 athymic nude mice and 5 mice
were treated with Avastin/Bevacizumab (10 mg/Kg) twice a
week, and the other 5 mice with Human IgG (10 mg/Kg). Mice
were sacrificed when tumors had reached a size of $2 cm per
IACUC protocol. Tumors were fixed in 1% PFA overnight then
allowed to sink completely in 50 mL of 30% Sucrose. They were
then frozen in blocks of OTC and sectioned at 20 mm.
Murine myocardial infarction. Myocardial infarction
utilizing a total occlusion model was induced in mice as described in
MDA-MB 231 breast Cancer Xenografts. Xenografts were
generated at Stanford University, approved by Stanfords
Institutional Animal Care and Use Committee and in accordance with
all Administrative Panel on Laboratory Animal Care (APLAC)
regulations at Stanford University and were in compliance with
the National Institutes of Health Guide for Care and Use of
Animals, and have been described in . Briefly, NOD/SCID
(non-obese diabetic severe combined immunodeficiency) female
mice had MDA-MB-231 breast cancer cells in 100 ml phosphate
buffered saline (PBS; pH 7.4) plus 100 ml of matrigel (BD
Biosciences, San Jose, CA, USA) injected into their left second
mammary fat pads. At 55 days after injection, when the average
tumor volume was 1.5 cm mice were euthanized for and tumors
excised and embedded in paraffin.
Neonatal hypoxic brain tissue. Following autopsy, brains
were immersed in phosphate buffered saline with 4%
paraformaldehyde for three days. On day 3, the brain was cut in the coronal
plane at the level of the Mamillary Body and immersed in fresh
4% paraformaldehyde/PBS for an additional three days. Post
fixation, all tissue samples were equilibrated in PBS with 30%
sucrose for at least 2 days. Following sucrose equilibration, tissue
was placed into molds and embedded with OCT for 30 to 60
minutes at room temperature or 4uC followed by freezing in dry
ice-chilled ethanol or methyl butane. The diagnosis of hypoxic
ischemic encephalopathy (HIE) requires clinical and pathological
correlations. With respect to the pathological features, all HIE
cases in this study showed consistent evidence of diffuse white
matter injury, including astrogliosis and macrophage infiltration.
These findings were confirmed by the increase in the number and
the staining intensity of GFAP- or CD68-positive cells, respectively
(not shown). In addition, HIE cases also showed evidence of
neuronal injury, including the presence of ischemic neurons and
variable degrees of neuronal loss, in cerebral cortex, hippocampus
and basal ganglia (not shown).
Adherent cells were washed twice by addition of ice cold PBS to
the monolayer and disposal of the supernatant. 1 ml of freshly
made ice cold lysis/wash buffer (50 mM Tris-HCl, 150 mM NaCl
pH 7.5, 1% Nonidet P40 0.5% sodium deoxycholatex
supplemented with 1 complete tablet from Roche) was added to the
washed cell monolayers to achieve a concentration of 106107
cells/ml. Cells were scraped into an eppendorf, and sonicated on
ice with 5 pulses each for 8 seconds long. Lysate was spun down at
13000 rpm for 5 minutes. Supernatant (except 200 ml) was put
onto a new tube. The un-lysed pellet was resuspended into the
200 ml remaining lysate, and sonicated again, the tube centrifuged
at 13000 rpm for 5 minutes and the new lysate added to the
original lysate. This was repeated three times until complete lysis
was achieved. 50 ml of this lysate was kept aside as input. To
reduce background a preclearing step was performed overnight.
50 ml of the homogeneous protein G- agarose (Roche) suspension,
equilibrated in the lysis buffer was added to the 1 ml lysate at 2
8uC on a rotating platform overnight. Beads were then pelleted by
centrifugation at 20006g for 2 minutes at 4uC. Supernatant was
transferred to a new tube. 50 ml of Chromotek-GFP-Trap bead
(Allele Biotechnology), a GFP-binding protein based on a single
domain antibody derived from Lama alpaca, was equilibrated in
the wash/lysis buffer, centrifuged for 2 minutes at 20006g, and
supernatant discarded. The cell lysate was added to these beads
and rotated (gentle end-over-end mixing) for 1 hour at 4uC. The
lysate/bead complex was then centrifuged for 2 minutes at
20006g. Pellet was washed 4x by resuspending in 1 ml lysis/wash
buffer. A final wash was performed once for 30 minutes by
endover-end mixing. Beads were then resuspended in 90 ml of 2x SDS
pro-track sample buffer (Lonza), boiled for 10 minutes at 95uC.
Beads were collected by centrifugation at 27006g for 2 minutes at
4uC and SDS-PAGE performed with the supernatant.
Conceived and designed the experiments: KA AR PFR EM. Performed the
experiments: KA AR VN TS HJC KVT AJ MD MD. Analyzed the data:
KA YY SSJ PFR HJC KVT DHR MA EM. Contributed reagents/
materials/analysis tools: YY SSJ DHR MA. Wrote the paper: KA EM.
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