Mitochondrial NDUFS3 regulates the ROS-mediated onset of metabolic switch in transformed cells

Article (PDF Available)inBiology Open 2(3):295-305 · March 2013with22 Reads
DOI: 10.1242/bio.20133244 · Source: PubMed
Aerobic glycolysis in transformed cells is an unique metabolic phenotype characterized by a hyperactivated glycolytic pathway even in the presence of oxygen. It is not clear if the onset of aerobic glycolysis is regulated by mitochondrial dysfunction and, if so, what the metabolic windows of opportunity available to control this metabolic switch (mitochondrial to glycolytic) landscape are in transformed cells. Here we report a genetically-defined model system based on the gene-silencing of a mitochondrial complex I subunit, NDUFS3, where we demonstrate the onset of metabolic switch in isogenic human embryonic kidney cells by differential expression of NDUFS3. By means of extensive metabolic characterization, we demonstrate that NDUFS3 gene silencing systematically introduces mitochondrial dysfunction thereby leading to the onset of aerobic glycolysis in a manner dependent on NDUFS3 protein levels. Furthermore, we show that the sustained imbalance in free radical dynamics is a necessary condition to sustain the observed metabolic switch in cell lines with the most severe NDUFS3 suppression. Together, our data reveal a novel role for mitochondrial complex I subunit NDUFS3 in regulating the degree of mitochondrial dysfunction in living cells, thereby setting a "metabolic threshold" for the observation of aerobic glycolysis phenotype within the confines of mitochondrial dysfunction.
Mitochondrial NDUFS3 regulates the ROS-mediated
onset of metabolic switch in transformed cells
Sonal Suhane
*, Hirotaka Kanzaki
*, Vaithilingaraja Arumugaswami
, Ramachandran Murali
V. Krishnan Ramanujan
Metabolic Photonics Laboratory,
Department of Surgery,
Department of Biomedical Sciences,
Regenerative Medicine Institute,
Imaging Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Davis 6066, Los Angeles, CA 90048, USA
*These authors contributed equally to this work
Author for correspondence (
Biology Open 2, 295–305
doi: 10.1242/bio.20133244
Received 1st October 2012
Accepted 6th December 2012
Aerobic glycolysis in transformed cells is an unique metabolic
phenotype characterized by a hyperactivated glycolytic
pathway even in the presence of oxygen. It is not clear if
the onset of aerobic glycolysis is regulated by mitochondrial
dysfunction and, if so, what the metabolic windows of
opportunity available to control this metabolic switch
(mitochondrial to glycolytic) landscape are in transformed
cells. Here we report a genetically-defined model system
based on the gene-silencing of a mitochondrial complex I
subunit, NDUFS3, where we demonstrate the onset of
metabolic switch in isogenic human embryonic kidney cells
by differential expression of NDUFS3. By means of extensive
metabolic characterization, we demonstrate that NDUFS3
gene silencing systematically introduces mitochondrial
dysfunction thereby leading to the onset of aerobic
glycolysis in a manner dependent on NDUFS3 protein
levels. Furthermore, we show that the sustained imbalance
in free radical dynamics is a necessary condition to sustain
the observed metabolic switch in cell lines with the most
severe NDUFS3 suppression. Together, our data reveal a
novel role for mitochondrial complex I subunit NDUFS3 in
regulating the degree of mitochondrial dysfunction in living
cells, thereby setting a metabolic threshold for the
observation of aerobic glycolysis phenotype within the
confines of mitochondrial dysfunction.
2013. Published by The Company of Biologists Ltd. This is
an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial Share Alike
License (
Key words: Mitochondrial complex I, NDUFS3, Metabolic switch,
Aerobic glycolysis, Reactive oxygen species, Mitochondrial
Cell transformation often entails alterations in energy metabolism
to accommodate adaptation in cellular functions as well as to
reprogram the interactions within the cellular environment
(Tennant et al., 2009). Emerging trend in metabolic profiling of
healthy and disease-related tissues has fundamental implications
in not only understanding the metabolic origins of the disease
phenotype but also in designing effective strategies for
prevention as well as treatment (Brandon et al., 2006; Unwin et
al., 2003; Wallace, 1999; Wang et al., 2009). In a normal
metabolic paradigm, cellular energy demands are primarily
met by oxygen-dependent mitochondrial metabolism of
carbohydrates, fatty acids and amino acids in almost all cell
types while oxygen-independent glycolysis accounts for only a
fraction of cellular energy supply. Global propagation of this
bioenergetics landscape, from single cells to tissues to the organs,
may critically depend on metabolic thresholds that would in turn
determine the limits of proper functioning of various tissues. For
instance, in the extreme case of hypoxia, anaerobic glycolysis
provides for cellular survival, owing to mitochondrial impairment
in the absence of oxygen (Hanahan and Weinberg, 2000;
Semenza, 2007). A counter-intuitive biochemical phenotype
often observed in transformed cancer cells is their increased
propensity to utilize glycolytic pathway even in the presence of
oxygen. This observation led to a hypothesis in the 1950s that this
feature (‘aerobic glycolysis’) could stem from acute
mitochondrial dysfunction in the cancer cells. In fact, the
glycolytic upregulation in tumors has been shown to correlate
with proliferative advantages, increased aggressiveness and poor
chemosensitivity (Chen et al., 2007; Gogvadze et al., 2010; Hu et
al., 2012; Mathupala et al., 2009; Vander Heiden et al., 2009;
Warburg, 1956). Despite the fact that this hypothesis has not been
proved unanimously, the feature has been long exploited in
clinical imaging modalities such as positron emission
tomography where tumor imaging contrast is usually achieved
by the preferential uptake of isotope-labeled glucose analogs by
the tumors (Fischman, 2008). Based on emerging experimental
evidences, the metabolic shift from mitochondrial to glycolytic
phenotype is no longer viewed as a compromise in cellular ATP
as was originally speculated but more as a basis of connecting the
increased glycolytic precursors to biosynthetic pathway for rapid
proliferation in cancer cells (Vander Heiden et al., 2009). Our
laboratory has been involved in addressing the question of
metabolic switch (i.e. mitochondrial pathway to aerobic
glycolysis pathway) from the mitochondrial point of view and
more specifically by investigating the role of mitochondrial
Research Article 295
Biology Open
complex I in facilitating the metabolic switch in transformed
cancer cells.
Mitochondrial complex I is the critical entry point of
mitochondrial electron transport chain. Electron donor, NADH,
from the TCA cycle is being oxidized by this huge multi-subunit
complex I, which then initiates the electron transport. In this
context, it is still not clear if mitochondrial dysfunction has a
causative role in the observed metabolic switch or if the observed
mitochondrial ramifications are simply a consequence of cellular
adaptation to an increased glycolytic phenotype (Bellance et al.,
2009; Brandon et al., 2006; Nijtmans et al., 2002; Pelicano et al.,
2009). A major bottleneck in addressing this vital question is the
absence of a genetically defined model system for systematic
modulation of mitochondrial function and to dissect the various
modes of achieving the metabolic switch in transformed cells. An
immediate significance of such a model system will be the
possibility to design effective strategies for controlled
perturbation of mitochondrial function as well as the possibility
of therapeutic modules for targeting critical sites of
mitochondrial network. Earlier, we reported that the protein
expression of a mitochondrial complex I precursor subunit
NDUFS3 was significantly higher in invasive breast carcinoma
tissues obtained from clinical patient specimens as compared
with normal breast tissues (Suhane et al., 2011). We further
discovered that this aberrant expression of NDUFS3 correlated
with hypoxic/necrotic regions of the tumor tissues indicating a
correlation with tumor aggressiveness. It is intriguing that one of
the subunits in the complex-I chain is deregulated in breast
cancer. It is unclear if the subunit NDUFS3 has an independent
function in regulating metabolic features of transformed cells.
We hypothesized that differential expression of NDUFS3 might
yield insights into the role of mitochondrial complex I assembly/
function in energy metabolism of transformed cells. To test this
hypothesis, we established stable cell lines (transformed human
embryonic kidney cells) by shRNA-mediated gene silencing
NDUFS3 expression and by characterizing the metabolic
phenotypes in these isogenic cells. Our data indicate that
differential NDUFS3 expression profoundly affects the overall
mitochondrial function and enable the cells to undergo metabolic
switch (mitochondrial to aerobic glycolysis). The observed
metabolic switch phenotype is reversible in cell lines with
modest suppression of NDUFS3 levels whereas it is irreversible
in cell lines with the most severe suppression of NDUFS3 levels.
Our experiments further point out to a critical role that the
reactive oxygen species (ROS) status plays in facilitating this
metabolic switch. In the absence of any genetically-defined
model system for studying the role of mitochondrial complex I
function in transformed cells, this study provides the first such
evidence based on a clinically-relevant mitochondrial complex I
subunit, NDUFS3 function.
Materials and Methods
Generation of stable cell lines expressing shRNA constructs
Human embryonic kidney (HEK-293T) cells were maintained in DMEM medium
(1 g/l glucose) supplemented with 10% fetal bovine serum and antibiotics. The
shRNA set (5 clones) containing lentiviral pLK0.1 vectors were purchased from
Thermo Scientific (catalog no. RHS4533; Openbiosystems, Brookfield, WI, USA).
Each shRNA against NDUFS3 target gene was packaged individually in HEK-
293T cells using VSV-G envelop pseudotyping. We also included a pLKO.1 vector
containing non-specific shRNA as control (sequence: CAA CAA GAT GAA GAG
CAC CAA). The packaged lentiviral particles were concentrated by
ultracentrification at 30,000 RPM at 4
C for 90 minutes in a Beckman Coulter
Optima LE-80K ultracentrifuge with a SW32 Ti swing bucket rotor. The pelleted
viral particles were resuspended in 100 ml of DMEM. The viral titer in transducing
units was measured in HEK-293T cells. 10-fold serially diluted viruses were
inoculated onto the cells in each well of a 96 well plate. Forty eight hours after
inoculation, puromycin antibiotic (5 mg/ml) was added to each well. 3–4 days post
antibiotic selection, puromycin resistant cell clones were counted and transducing
units per ml of each viral vector was calculated. For establishing cell lines
constitutively expressing shRNA, the cell lines were plated in a 48-well plate.
Individual viral vectors were inoculated onto cells in each well. The next day viral
inoculum was replaced with fresh cell culture medium. Stable cell lines were
established followed by puromycin antibiotic selection.
Quantitative real-time PCR
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA)
according to instruction of manufacturer. The amount and the integrity of RNA
were assessed by measurement of absorbance at 260 and 280 nm. Total RNA was
reverse transcribed into first strand cDNA using iScript cDNA synthesis kit (Bio-
Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer.
Quantitative real-time PCR was performed with SsoFast EvaGreen (Bio-Rad
Laboratories, Inc.). Primer sequences (Integrated DNA Technologies, Skokie, IL,
USA) were as follows:
TG-39 and
reverse 59-GGC CAG GTG AAT ATG TTT AG-39; HK2 forward,59-
reverse 59-ACA GTG CAC ACC
39 and
reverse 59-ACG CTG AGC CAG TCA GTG TA-39. CT value was
normalized to the CT value of 18S ribosomal 1 (RN18S1) rRNA in the same
sample. Relative expression changes of mRNA between shNDUSF3 expressed
cells and control shRNA expressed cells were calculated by using DDCT method.
The comparisons of mRNA expression levels were conducted using the DDCT
method, where the DDCT was the difference in the DCT values between two
samples and 2
represents the fold change in mRNA expression.
Immunoblotting and immunofluorescence
Cells were lysed in radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM
Tris-HCl (pH 8.0), 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS) (Sigma–Aldrich, St. Louis,
MO, USA) containing protease inhibitor cocktail and phosphatase inhibitor
cocktail 2. Cell lysates were centrifuged at 15,000 g for 10 minutes at 4
C, and
protein concentrations were determined by DC protein assay reagent (Bio-Rad
Laboratories, Inc., Hercules, CA, USA). Western blot analysis was performed
according to the guidelines of Trans-Blot Turbo Transfer system protocol. In brief,
30 mg of total proteins were heated for 5 minutes at 95
C, and then separated on 4–
20% SDS-polyacrylamide gel and electrotransferred to nitrocellulose membrane.
Visualizing protein band was performed according to the guidelines of SNAP i.d.
standard protocol (EMD Millipore, Billerica, MA, USA). Membranes were
blocked in 0.25% of non-fat dry milk in Tris-buffered saline-Tween 20 (TBS-T)
buffer, and then incubated with the following primary antibodies: 1:2000 mouse
monoclonal anti-NDUFS3 (ab110246, Abcam, Cambridge, MA, USA), 1:2000
mouse monoclonal anti-NDUFA9 (ab14713, Abcam, Cambridge, MA, USA),
1:1000 rabbit monoclonal anti-HK2 (no. 2867, Cell Signaling Technology, Inc.,
Dancers, MA, USA), 1:1000 rabbit polyclonal anti-PKM2 (no. 3198, Cell
Signaling Technology, Inc., Dancers, MA, USA). The following four rabbit,
polyclonal antibodies were purchased from Abgent Inc., San Diego, CA, USA:
NDUFS1 (Ap5678c, 1:100), NDUFS2 (Ap9769c, 1:100), NDUFS4 (Ap6932b,
1:100) and NDUFS8 (Ap12552c, 1:100). The Mitoprofile OxPhos westernblot
cocktail (ab110411, Abcam, Cambridge, MA, USA) contained a stoichiometric
mixture of five mouse monoclonal antibodies specifically targeting the following
subunits: complex I subunit NDUFB8 (20 kDa), complex II subunit (30 kDa),
complex III subunit Core 2 (47 kDa), complex IV subunit II (24 kDa) and ATP
synthase subunit alpha (55 kDa). After washing with TBS-T, membranes were
incubated with peroxidase conjugated secondary antibody in TBS-T. Blots were
washed and hybridization signals were measured by enhanced chemiluminescence
detection system using Luminata Forte Western HRP Substrate (EMD Millipore,
Billerica, MA, USA).
For immunofluorescence experiments, HEK Ctrl and NDUFS3-deficient cell
lines were seeded on 12 mm glass coverslips in 24 well plates (80,000–120,000
cells/well) for 24 hours. Next day, cells were washed with PBS twice followed by
fixation with 4% paraformaldehye/PBS for 20 minutes at RT and then cells were
washed three times with PBS. Fixed cells were heated in antigen-retrieval buffer
(100 mM Tris, 5% (w/v) urea, pH 9.5) at 95
C for 10 minutes followed by
permeabilization with 0.1% Triton X-100/PBS for 15 minutes at RT and then
washed three times with PBS. 10% goat serum in PBS was used for blocking for
1 hour at RT and then overnight incubation was done with mouse NDUFS3 (1:150,
catalog no. ab110246; Abcam, Cambridge, MA, USA) and Hexokinase 2 (1:1600,
catalog no. 2867; Cell Signaling Technology, Inc., Dancers, MA, USA) at 4
Next day, cells were washed three times with 1% goat serum in PBS at RT for
10 minutes each wash and then incubated with appropriate secondary antibody in
10% goat serum in PBS (Rabbit Alexafluor488, 1:400, catalog no. A11034;
Alexafluor594, 1:400, catalog no. A11032; Life Technologies (Invitrogen), Grand
Mitochondrial NDUFS3 296
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Island, NY, USA) at RT for 2 hours and then washed three times with 1% goat
serum in PBS for 10 minutes each wash at RT. Cells were counterstained with
300 ng/ml DAPI in 1% goat serum (catalog no. D3571; Life Technologies
(Invitrogen), Grand Island, NY, USA) for 10 minutes and then washed with PBS
for 1 minute. Coverslips were mounted with prolong DAPI (catalog no. P36935;
Life Technologies (Invitrogen), Grand Island, NY, USA) as described earlier
(Suhane and Ramanujan, 2011). Images were acquired in a Leica SP5 confocal
microscope with spectral detector.
Metabolic characterization
Flow cytometry analysis
Flow cytometry analysis of live cells was performed in FACScan flow cytometer
(BD Biosciences) with appropriate labeling mix in the cell population: 100 mM
2NBDG (Life Technologies (Invitrogen), Grand Island, NY, USA; glucose
uptake), 2.5 mM DCFDA (Life Technologies (Invitrogen), Grand Island, NY,
USA, hydrogen peroxide levels), 2.5 mM MitoSox Red (Invitrogen, superoxide
levels), 200 nM TMRM (Life Technologies (Invitrogen), Grand Island, NY, USA,
mitochondrial membrane potential). Apoptotic cell population was determined by
labeling live cells with a polarity sensitive indicator of viability and apoptosis
(10 ml/ml working concentration; pSIVA-IANBD, Imgenex, San Diego, CA, USA)
30 minutes in PBS with Ca
before flow cytometry.
Live cell kinetics imaging
Real-time metabolic response kinetics was monitored in live cells in Delta T
chambers (Bioptechs, Butler, PA, USA) and imaging with a two-photon excitation
microscope (Leica SP5 MP) built around a femtosecond pulsed laser (Mai Tai,
Spectra Physics, Santa Clara, CA, USA). A typical time lapse consisted of
collecting images (acquisition time ,3 seconds/image) every 20 seconds for about
40 minutes.
Lactate measurements
Typically 4 million cells of control and the shRNA clones were plated in 100 mm
dishes in complete medium. Extracellular lactate generation was measured using a
commercial Lactate Plus analyzer (Sports Resource, USA) by allowing the cell
cultures to grow for 36–48 hours in 100 mm petri dishes in complete DMEM
medium. Cumulative lactate content was measured at the end of the incubation
period. The lactate concentration of the complete medium without cells was found
to be 1.4 mM and this value was subtracted from the experimental values
measured for the three cell lines before normalizing with respect to the total cell
number in each case and the cell culture duration. The final values of lactate
generation rate (millimole/million cells/hour) were used to compare the differences
between the control and the NDUFS3-deficient ShRNA clones.
Oxygen consumption measurements
Mitochondrial oxygen consumption [pO
] data were obtained with a clark-type
oxygen microelectrode (Strathkelvin Instruments, Scotland) in a closed-cell
respirometry design. The probe electrodes were calibrated with 5% sodium
thiosulfite solution (0% oxygen) and mammalian ringer solution (100% oxygen
,230 mmol/l) at 38
C. All measurements were performed either in PBS or cell
culture medium. Oxygen consumption rate was then calculated from the raw pO
data as described earlier (Suhane and Ramanujan, 2011).
Amplex Red assay
Supporting measurements for evaluating ROS status in the HEK control and the
NDUFS3-deficient shRNA clones were carried out by using Amplex Red assay
(catalog no. A22188; Life Technologies (Invitrogen), Grand Island, NY, USA).
Briefly this assay relies on the enzymatic activity of hydrogen peroxide/peroxidase
pair measured by the Amplex Red reagent (10-acetyl-3-7-dihydroxyphenoxazine).
In the presence of peroxidase, the Amplex Red reagent reacts with H
in a 1:1
stoichiometry to produce the red-fluorescent oxidation product, resorufin (571 nm
excitation; 585 nm emission). HEK Control and shRNA
and shRNA
were plated in 12 well plates (300,000 cells/well). After 36 hours of culture, the
cells were trypsinized and resuspended in 20 ml KRPG buffer and then added to
the 100 ml of the reaction mixture. The reaction mixture consisted of 50 mM
Amplex Red Reagent and 0.1 U/ml HRP in Krebs-Ringer phosphate (145 mM
NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mM CaCl
, 1.22 mM
, 5.5 mM glucose in deionized water, pH 7.35). After incubating the
mixture for 30 minutes at room temperature, resorufin fluorescence was measured
in a microplate reader. The system was calibrated with hydrogen peroxide
standards prior to the experiment to obtain a standard curve. The final readings
were converted to the net generated H
values based on this standard curve. All
experiments were done in triplicates.
Mitochondrial complex I enzyme activity assay
Cultured cells were washed three times with cold PBS followed by lysis with
sonication in cold PBS and then centrifuged at 10,000 g for 10 minutes at 4
Supernatant was collected and protein concentration was determined. Complex I
activity was measured by following the decrease in absorbance due to oxidation of
NADH to NAD at 360 nm. Absorbance measurements were carried out in a
microplate reader (Infinite 200 Pro, Tecan Group Ltd, Switzerland). Instrument
linearity was first checked with serial dilution of NADH stocks (50–200 mg
NADH) and the data acquisition time was optimized to minimize photobleaching
artifacts. The reaction mixture contained 25 mM potassium phosphate, pH 7.4,
5 mM MgCl
, 5 mM KCN, 2.5 mg BSA, 100 mM NADH and 400 mM
ubiquinone. 200 mg of cell lysates of HEK control, shRNA
and shRNA
cell lines were first added to independent wells in the microplate reader along with
the above reagents (total reaction volume 100 ml) and the absorbance kinetics
measurement was initiated. After monitoring absorbance from these wells for
,4 minutes (20-second intervals), 2 mM rotenone was added to the three wells to
monitor the rotenone-sensitive absorbance kinetics. Initial rates of NADH
oxidation before and after rotenone addition were computed by linear regression
analysis. Rotenone-sensitive complex I activity was then calculated by subtracting
the rotenone-insensitive component from the total activity (before rotenone
addition) and is presented as complex I specific enzyme activity per mg protein per
minute. The data presented in this paper are a representative of three independent
Data presented are mean6s.e. from at least three independent experiments unless
otherwise mentioned. Statistical significance was estimated based on the Student’s
t-test (P,0.05).
Generation of HEK cell lines with differential NDUFS3
Human embryonic kidney (HEK-293T) cells were transduced
with five lentiviral constructs as described earlier. Real-time PCR
measurements revealed that all clones except one showed
significant suppression of NDUFS3. This observation of partial
reduction in NDUFS3 levels in different clones provided an
unique opportunity to evaluate the distinct role of NDUFS3
stoichiometry in determining the metabolic phenotypes resulting
from the differential suppression of NDUFS3 in the transformed
cells. For the sake of clarity and in order to demonstrate the role
of NDUFS3 expression level in the observed metabolic
phenotype, in this paper we have chosen to present results of
metabolic characterization obtained from only two shRNA
clones. Fig. 1 shows the relative mRNA levels in the control
and the NDUFS3-deficient shRNA clones. Immunoblotting of
cell homogenates further revealed that showed robust suppression
at the protein level (Control: 100%; shRNA
: 73%; and
: 33%). The observed discrepancy between mRNA
levels and the protein levels in the shRNA clones is not
uncommon given the fact that starting with a certain mRNA
level, multiple regulatory steps could control final protein
expression levels. Since the protein level is physiologically
relevant for interpreting the metabolic phenotype in cells, all the
results presented in this paper refer to these two cell lines with
aforementioned suppression levels. High resolution imaging of
single cells clearly indicated the mitochondrial distribution of
NDUFS3 in all the cell lines as shown in the representative
Fig. 1b. Currently available mitochondrial complex I assembly
models suggest that NDUFS3 is one of the earliest precursor
subunits (Koopman et al., 2010). A preliminary search for
potential interacting partners of NDUFS3 yielded two candidates,
NDUFS2 and NDUFS8, which are in the same Fe–S cluster of
mitochondrial complex I ( Immunoblotting
for these two proteins in the control and NDUFS3-deficient cell
lines showed no detectable changes due to NDUFS3 suppression.
However, two other Fe–S cluster subunit levels (NDUFS1 and
NDUFS4) were found to be altered in NDUFS3-deficient cell
lines (Fig. 1c). In order to verify that the NDUFS3 suppression
did not significantly alter the assembly of later stages of
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mitochondrial complex I thereby altering the overall function of
this complex, we monitored the protein levels of a downstream
assembly subunit NADH dehydrogenase [ubiquinone] 1 alpha
sub complex subunit 9 (NDUFA9) in the control and the two
NDUFS3-deficient cell lines (Fig. 1c). NDUFS3 suppression did
not significantly alter the NDUFA9 levels in these cell lines.
Under the same cell culture conditions, the shRNA
cells showed reduced growth rates as compared with
the control cells. Cell cycle analysis in these three cell lines is
summarized in Fig. 2a,b. We observed that both the stable cells
had relatively larger S-phase (DNA synthesis phase) population
in comparison with the control cell lines. However, flow
cytometry analysis of cell proliferation (using CFSE labeling)
in these three cell lines showed a reduction in proliferation
exponent (Fig. 2c). We hypothesized that such a reduction in
proliferative potential might be related to increase in DNA-
synthesis population in the shRNA cells, which could be
reconciled by monitoring the basal apoptotic activity in these
cells. Using a sensitive apoptosis probe in living cells, we further
confirmed that the NDUFS3 shRNA
and shRNA
have higher basal apoptotic activity (2-fold and 3-fold,
respectively) than control cell lines, which accounted for the
apoptosis-related increase in S-phase (DNA fragmentation)
NDUSF3 silencing induces mitochondrial dysfunction in HEK
Mitochondrial electron transport chain function critically depends
on proper assembly and function of multiple subunits and
disrupting this balance by modulating the expression level of one
of the precursor subunits such as NDUFS3 is expected to affect
the mitochondrial function. Since our goal in this study was to
evaluate the effects of such a controlled perturbation of the net
mitochondrial function, we developed a set of sensitive assays for
evaluating the effects of silencing NDUFS3 expression:
expression levels of OxPhos complex systems, mitochondrial
depolarization, oxygen consumption and oxygen consumption
rate (OCR). Fig. 3a,b show representative mitochondrial complex
I activity in the control and the NDUFS3-deficient cell lines. The
parental cells displayed relatively higher NADH oxidation rate
indicating robust complex I activity whereas NDUFS3
suppression systematically reduced mitochondrial complex I
activity in shRNA
and shRNA
cells. A sensitive indicator
of mitochondrial function is the mitochondrial membrane
potential, which reports the potential difference between the
mitochondrial matrix and the intermembrane space during
metabolic activity. Any perturbation to mitochondrial function
will affect the membrane potential and the integrity of
mitochondrial function can be tested by the rate of
mitochondrial membrane depolarization (collapse of the
membrane potential) in living cells. In order to compare the
integrity of mitochondrial function in the control and the two
stable cells, we labeled the live cells with membrane permeable
mitochondrial membrane potential marker (TMRM) as described
in Materials and Methods. As established in earlier studies
(Genova et al., 1997; Ramanujan and Herman, 2007), we
employed mitochondrial complex I inhibitor (100 nM rotenone)
as a perturbation reagent, to elicit real-time metabolic responses
in TMRM-labeled live cells and monitored the kinetics by two-
photon excitation microscopy (Ramanujan and Herman, 2008;
Ramanujan et al., 2008; Ramanujan et al., 2005). The rotenone-
dependent mitochondrial depolarization kinetics was observed to
be drastically reduced in the shRNA
and shRNA
cells in
contrast to the rapid depolarization kinetics in the control cell
lines (Fig. 3c,d). A semi-quantitative analysis of initial decay
rates showed that the complex I-inhibition-induced mitochondrial
depolarization rates were as follows: control cells
; shRNA
cells (0.2860.03)6min
; and
Fig. 1. Generation of human embryonic kidney (HEK) cells with selective gene silencing of mitochondrial NDUFS3. (a) Relative mRNA expression level of
NDUFS3 in control and the two NDUFS3-deficient (shRNA
and shRNA
) cells as measured by real-time PCR assay. The data are shown after normalizing with
the expression of 18S rRNA in each cell line. The inset shows a representative immunoblot result obtained from cellular homogenates. NDUFS3 band intensities were
normalized with respect to those of beta actin bands in each case and shown as the percentage of the control cell line. The two clones that are described in this paper
had protein expression levels as follows: shRNA
: 73% (27% suppression) and shRNA
: 33% (67% suppression). (b) Representative high-resolution confocal
image of HEK control cell lines shown here were paraformaldehyde fixed and labeled with primary antibody against NDUFS3 (mouse-NDUFS3) and a fluorescent
Alexa594 conjugated secondary antibody (anti-mouse-Alexa594) for immunofluorescence visualization. Similar mitochondrial distribution of NDUFS3 (shown in
green) and nuclear DAPI (shown in red) was seen in all the three cell lines. Scale bar520 mm. (c) Representative immunoblot results of four Fe–S cluster subunits
(NDUFS1, NDUFS2, NDUFS4 and NDUFS8) as well as that of a late assembly subunit NDUFA9 probed in cellular homogenates to verify the effect of NDUFS3
suppression on the related complex I subunit expression levels.
Mitochondrial NDUFS3 298
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cells (0.1060.02)6min
. In order to verify these
mitochondrial effects at the overall functional level in live cells,
oxygen consumption measurements in NDUFS3 shRNA
cells showed that there was a net reduction in oxygen
consumption rate (Fig. 3e,f) in these cell lines as compared with
the control cells. These results, together with the mitochondrial
membrane depolarization data, further confirmed that NDUFS3
silencing induced mitochondrial dysfunction in live cells in a
manner dependent on the NDUFS3 expression level. Fig. 3g
shows immunoblotting result where we simultaneously
monitored the representative subunits of all the five complexes
of the mitochondrial oxidative phosphorylation (OxPhos) system.
NDUFS3 suppression was correlated with a concomitant
upregulation of complex III and V subunits revealing
compensatory effects and/or mitochondrial stress.
NDUFS3-silencing induced mitochondrial dysfunction
exacerbates aerobic glycolysis phenotype
As described in the earlier section, there is still no clarity on the
causative role of mitochondrial dysfunction in the aerobic
glycolysis phenotype observed in transformed cells. In the
present study where we have established a genetically defined
model system of NDUFS3 silencing, we sought out to evaluate
the role of resulting mitochondrial dysfunction in the observation
of aerobic glycolysis. Fig. 4a shows the average (three
independent experiments) glucose uptake rate in the three cell
lines under investigation. Both the shRNA
and shRNA
cells displayed a modest increase in glucose uptake as compared
with the control cells. An established hallmark of aerobic
glycolysis is the increased rate at which lactate is generated
owing to the fact that a larger proportion of pyruvate is converted
to lactate rather than entering the mitochondrial TCA cycle
(Suhane and Ramanujan, 2011). As can be seen from Fig. 4b,
cell line displayed nearly 3-fold higher lactate
generation rate as compared with control cell lines while
clone showed only a modest increase. This is an
intriguing finding given the fact that both the NDUFS3-deficient
cell lines displayed a reduction in mitochondrial function. It is
important to note here that, besides glucose metabolism, the
cellular lactate generation could result from other metabolic
pathways such as glutaminolysis. Since the major focus of this
study is to address the role of mitochondrial dysfunction in the
onset of aerobic glycolysis, no further attempts were taken to
characterize these alternate metabolic pathways and we utilized
lactate generation rate as a surrogate marker of aerobic glycolysis
phenotype. From the molecular point of view, earlier studies have
shown that aerobic glycolysis phenotype was accompanied by
upregulation of a key glycolytic enzyme hexokinase 2 (HK2) that
is mitochondrial outer membrane-bound in almost all cell types
(Mathupala et al., 2009). HK2 catalyzes the first committed step
of glycolysis (Fig. 5a). Real-time PCR and immunofluorescence
measurements revealed an increase in this enzyme level in
and shRNA
cells, both at the mRNA and the
protein levels supporting the observed increase in aerobic
glycolysis phenotype (Fig. 5b–d). A possible reason for an
increase in aerobic glycolysis in transformed cells is to divert the
Fig. 2. Characterization of cell growth and proliferation features in HEK cells. (a) Flow cytometry based cell cycle analysis measured by labeling the fixed cells
with propidium iodide (PI). Cell cycle analysis was performed by ModFit software (BD Biosciences, San Jose, CA, USA) and the cell populations in G0–G1, S and
G2–M phases are given in b.(c) Cell proliferation as measured by flow cytometry after labeling with the CFSE probe and culturing for 72 hours. Cell proliferation
exponent indicates the differential cell growth/proliferation characteristics in the NDUFS3-deficient cell lines as compared with the control cell lines. (d) Basal
apoptotic rate was measured by labeling live cells with pSIVA-IBND that labels the exposed phosphatidyl serine in early stages of apoptosis in each cell line.
(* denotes significant difference, P,0.05).
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glycolytic precursors to biosynthetic pathway by inhibiting the
conversion of phosphoenol pyruvate to pyruvate (catalyzed by
M2 isoform of pyruvate kinase, PKM2) (Christofk et al., 2008)
(Fig. 5a). In our case, we did not find any observable difference
in PKM2 levels in all the three lines studied, thereby confirming
no direct role in biosynthetic pathway (Fig. 5c). This is in
Fig. 3. Measurement of mitochondrial parameters in the NDUFS3-deficient cell lines. (a) Mitochondrial complex I enzyme activity as measured by the rate of
oxidation of NADH (substrate) to NAD
by the complex I enzyme in the total cell lysate. Absorbance at 340 nm (NADH absorption maximum) was monitored after
adding the substrate (NADH) in the presence of the cell lysate and after ,4 minutes, 2 mM rotenone (complex I inhibitor) was added to monitor the rotenone-
sensitive NADH oxidation rates. Complex I specific NADH oxidation rates are summarized in b. In consensus with the mitochondrial dysfunction scenario, the
shRNA clones showed much delayed mitochondrial membrane depolarization rates as compared with the control cell lines under the perturbation of mitochondrial
complex I inhibitor (100 nM rotenone). Rotenone-sensitive mitochondrial depolarization is an established means of monitoring mitochondrial integrity or
mitochondrial dysfunction as in the case of the NDUFS3-deficient cell lines (c,d). Concomitant reduction in overall mitochondrial oxygen consumption in the
NDUFS3-deficient cells further confirm that these cell lines have impaired mitochondrial function induced by NDUFS3 suppression (e,f). Expression levels of
representative subunits from each of the mitochondrial oxidative phosphorylation (OxPhos) complexes in control HEK cells and the NDUFS3-deficient isogenic cell
lines as assayed by using a OxPhos cocktail described in Materials and Methods. As can be seen there was a significant upregulation of complex III and V subunits in
the shRNA clones indicating compensatory adaptive increase in mitochondrial OxPhos subunits and/or mitochondrial stress (g).
Fig. 4. Mitochondrial dysfunction in NDUFS3-deficient
cell lines induces aerobic glycolysis in a NDUFS3-
dependent manner. (a) Average glucose uptake rate and
(b) average lactate generation rate in the HEK control and
NDUFS3-deficient cells demonstrate the onset of aerobic
glycolysis in the latter. Data shown are mean6s.e. from
three independent experiments and were normalized respect
to the values for the control cell lines. Statistical
significance was estimated for dataset with a difference in
mean values is within P,0.05 threshold. (* denotes
significant difference, P,0.05 and NS denotes
Not Significant).
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accordance with the cell cycle/proliferation data (Fig. 2), further
demonstrating that the observed increase in aerobic glycolysis
phenotype did not give the NDUFS3-deficient cells any
proliferative advantage.
Aerobic glycolysis observed in the NDUFS3-deficient cells is
regulated by the ROS imbalance
In order to further characterize the metabolic effects of NDUFS3-
silencing and to understand the putative mechanisms of the
observed metabolic switch (mitochondrial to aerobic glycolysis),
we carried out extensive measurements of free radical dynamics
in these cell lines as summarized in Figs 6–8. Flow cytometry
analysis of basal status of reactive oxygen species (ROS)
indicated that the shRNA
cells displayed a significantly
higher population (24%) of cells having a higher superoxide
signal as compared with the control and the shRNA
(,10%). Superoxide-selective (Mitosox labeling) and hydrogen
peroxide-selective (DCFDA labeling) signals were significantly
higher only in the shRNA
cells but not in shRNA
(Fig. 6b,c). This was further substantiated by real-time metabolic
responses in live cells as measured by the two-photon excitation
imaging of superoxide generation after a brief exposure to
mitochondrial complex I inhibitor (100 nM rotenone). As shown
in Fig. 6d, shRNA
cells displayed a sustained increase in
superoxide-selective Mitosox red signal in contrast to the control
and shRNA
cells. An increase in net apoptotic cells was
observed upon exposure of the cells to exogenous oxidative stress
(Fig. 6e). In order to further understand if the observed metabolic
responses to oxidative stress is reflected in the antioxidant status
of the cells, we measured the levels of two major antioxidant
enzymes, SOD2 (mitochondrially localized superoxide dismutase
2 catalyzing the conversion of superoxide to hydrogen peroxide)
and catalase (cytosolic enzyme converting hydrogen peroxide to
hydroxyl radical) under the conditions of complex I inhibition
(100 nM rotenone, 48 hours) and complex III inhibition (100 nM
Antimycin A, 48 hours). The rationale for this design was to
investigate the role of two known sites of ROS generation
(complex I and complex III) in contributing to the ROS
phenotype in the three cell lines (Ramanujan and Herman,
2007). Fig. 7 summarizes these results. shRNA
cells displayed a higher basal levels of antioxidant
status as compared with the control cell lines indicating a
constitutive mitochondrial dysfunction stemming from NDUFS3
suppression in these cell lines. However, under the various
perturbation conditions as described above, shRNA
cells displayed opposite trends of antioxidant status
(Fig. 7b,d). Integrity of mitochondrial matrix and electron
transport chain requires that in response to oxidative stress,
antioxidant machinery will correspondingly increase in order to
nullify the stress as was observed in the control and the
cells. On the other hand, shRNA
cells displayed
an opposite trend. This anomalous feature can be reasoned as
either due to the irreversible loss of mitochondrial integrity as a
whole and/or degradation of antioxidant proteins in these cell
Fig. 5. Aerobic glycolysis phenotype observed in NDUFS3-deficient cell lines is substantiated by aberrant hexokinase 2 expression. (a) Schematic of critical
molecular players involved in routing glycolytic oxidation product pyruvate to mitochondrial metabolism. Hexokinase 2 (HK2) catalyzes the first committed step of
glycose phosphorylation thereby initiating the conversion of 6-carbon glucose to 3-carbon pyruvate. The terminal step of pyruvate formation is catalyzed by the rate-
limiting enzyme, pyruvate kinase M2 (PKM2). Hyperactive PKM2 could inhibit the pyruvate formation thereby diverting the glycolytic precursors to biosynthetic
pathway. (b) Relative mRNA levels of hexokinase 2 were found to be significantly higher in both the shRNA
and shRNA
cell lines, which is further confirmed
at the protein level (c). (* denotes significant difference, P,0.05 and NS denotes Not Significant). Immunofluorescence labeling of cells plated on glass coverslips
was carried out with anti-HK2 antibody and Alexa 488-tagged secondary antibody and subsequent fluorescence imaging of these cells revealed a clear upregulation of
HK2 in NDUFS3-deficient cell lines (d(i),(iii),(v)). Also shown are the surface plots demonstrating a drastic increase in HK2 expression levels in NUDFS3-deficient
cell lines in accordance with immunoblotting results (d(ii),(iv),(vi)). No significant difference was observed in the PKM2 level (c) thereby suggesting no differences
in biosynthetic machinery are operative in the NDUFS3-deficient cell lines in comparison with the control HEK cells. Scale bars520 mm.
Mitochondrial NDUFS3 301
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lines. To test these possibilities, we treated the cells with
pharmacological agents that mimic cellular antioxidants (Fig. 8).
Cell permeable superoxide mimetic (MnTBAP) treatment indeed
had an effect on reducing the net cellular ROS signal in a
concentration-dependent manner as measured by Amplex Red
assay (Fig. 8a) and flow cytometry analysis (Fig. 8d) in the
control and the shRNA
cells. On the other hand, this
antioxidant mimetic did not have any significant effect on the
ROS levels of shRNA
cells. Furthermore, cytosolic
glutathione precursor, N-acetyl cysteine (NAC) did not show
any concentration-dependent reduction in ROS level (Fig. 8b)
indicating the source of oxidative stress is predominantly of
mitochondrial origin. More importantly, the superoxide mimetic
MnTBAP treatment was shown to reduce the net lactate
generation in control and the shRNA
cells but not in
cells further supporting the conclusion that
sustained ROS is not only a sufficient but a necessary
condition for the observation of aerobic glycolysis in the
cells. Together these data point to a scenario in
cells where the mitochondrial dysfunction induced by
the partial silencing of NDUFS3 was below a metabolic threshold
necessary to cause a ‘constitutively active’ metabolic switch
from mitochondrial to aerobic glycolysis. On the other hand, in
cells, a greater loss of NDUFS3 led to a, plausibly,
irreversible status of mitochondrial dysfunction where sustained
ROS imbalance was just not sufficient to exacerbate the aerobic
glycolysis phenotype but also necessary to sustain this phenotype
even in the presence of exogenous antioxidant mimetics.
The major goal of this study was to establish a genetically
defined model system for studying the role of mitochondrial
complex I in modulating metabolic switch in transformed cells.
Alterations in energy metabolism have been known to be the
common denominator for various disease models including
diabetes, obesity, cancer and aging. Owing to the central
importance of mitochondrial metabolism in cell survival and
apoptosis, any fundamental insight into the various mechanisms
by which mitochondrial dysfunction dictates the onset of disease
phenotype can have high significance. In this context, the present
study addresses a fundamental question of how subtle
mitochondrial dysfunctions have causative role in the metabolic
switch in transformed cells. As the term switch implies, it will
be intriguing to understand if the switch from predominantly
Fig. 6. Basal reactive oxygen species (ROS) data in the NDUFS3-deficient cell lines. (a) Representative flow cytometry histograms showing the basal superoxide
(red lines) and hydrogen peroxide (green lines) signals as measured by labeling the live cells with superoxide selective Mitosox Red (2.5 mM, 20 minutes, 37
C) and
hydrogen peroxide-selective DCFDA (2.5 mM, 20 minutes, 37
C) probes. (b,c) Statistical analyses of steady-state ROS status in the control and NDUFS3 deficient
cell lines obtained from at least three independent experiments (P,0.001 and NS denotes Not Significant). (d) Live cell kinetics imaging analysis of real-time
superoxide generation rate measured in live cells pre-labeled with 2.5 mM Mitosox Red. The time-lapse imaging was initiated after adding 100 nM rotenone
(mitochondrial complex I inhibitor) to monitor the superoxide generation kinetics. Fluorescence values in each case were normalized with respect to the fluorescence
at t50 seconds and presented as percentage change in fluorescence for the three cell lines studied. These kinetic profiles were representative of three independent
experiments under identical imaging conditions. (e) Apoptotic cell population determined under exogenous generic ROS inducer, hydrogen peroxide (25 nM H
14 hours, 37
C) showed similar response trend in the control and shRNA
cell lines whereas the shRNA
cell lines had constitutively higher apopotic cell death
even without treatment further substantiating the effects of sustained oxidative stress in these cell lines. Mitochondrial complex I inhibitor (100 nM rotenone,
14 hours, 37
C) was also used in this experiment to demonstrate that apoptotic rates could be regulated by ROS status independent of the source of oxidative stress.
Mitochondrial NDUFS3 302
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mitochondrial to predominantly glycolytic, even in the presence
of aerobic conditions, can be reversed or reprogrammed.
Answering this question has huge clinical potential as well,
since it is then possible to devise strategies to modulate the
metabolic switch and plausibly revert the so-called
‘dysfunctional mitochondria’ to normal-like mitochondrial
status. We believe that the present study is an important step
towards this direction where we have unraveled the role of a
mitochondrial complex I subunit NDUFS3 expression in
regulating the ROS-mediated aerobic glycolysis in transformed
isogenic HEK cells.
First of all, our extensive metabolic characterization of the
NDUFS3-deficient cells revealed that it is possible to
systematically induce mitochondrial dysfunction by gene
silencing NDUFS3 expression, which is known to be one of the
precursor subunits in the mitochondrial complex I assembly
(Janssen et al., 2006; Koopman et al., 2010; Vogel et al., 2007).
Currently efforts are underway in our laboratory for identifying
other critical subunits in the complex I by which it is possible to
exert control on the mitochondrial complex I assembly process.
Although the dependence of mitochondrial function on this
critical subunit (NDUFS3) is intuitive, there have been no
attempts to establish a model system without the complications of
hypoxia, as is done in the present study. Furthermore, the
mitochondrial dysfunction observed in the NDUFS3-deficient
cells seems to exacerbate the aerobic glycolysis thereby
confirming the role of mitochondrial complex I modulation in
metabolic switch in the transformed cells. Even though NDUFS3
suppression did not directly alter the potential NDUFS3-
interacting partners (NDUFS2 and NDUFS8) as well as the late
assembly subunit NDUFA9 (Fig. 1c), we can not exclude the
possibility that the observed metabolic phenotype in NDUFS3-
deficient cell lines could stem from a compromised complex I
assembly. Our observations of reduced complex I enzyme
activity (Fig. 3a) and alterations in other Fe–S cluster subunits
(Fig. 1c) support this possibility. On the other hand, despite the
fact that the two cell lines (shRNA
and shRNA
investigated in this study displayed similar mitochondrial
dysfunction in terms of complex I enzyme activity, oxygen
consumption, and the OxPhos subunit expression, the
exacerbated aerobic glycolysis phenotype was only observed to
be permanent (irreversible) in the cell lines with most sustained
Fig. 7. Antioxidant status in the control and the NDUFS3-deficient cell lines under various perturbation conditions. Basal antioxidant levels were measured
under three specific conditions of ROS induction. Mitochondrial complex I and complex III sites are known to be critical for mitochondrial ROS generation and
inhibition of these complexes is anticipated to elicit antioxidant responses in cells with intact mitochondrial function. Antioxidant enzyme levels (superoxide
dismutase, SOD2 and catalase) measured under (a,b) mitochondrial complex I inhibition (100 nM rotenone, 48 hours) and (c,d) mitochondrial complex III inhibition
(100 nM Antimycin A, 48 hours). Please refer to the main text and Fig. 5a for more details.
Mitochondrial NDUFS3 303
Biology Open
ROS imbalance (shRNA
cells). This points to an important
fact that there may exist a metabolic threshold for NDUFS3
stoichiometry in the overall mitochondrial complex I assembly/
function. One interesting approach to delineate the distinct roles
of metabolic regulatory function and the complex I assembly
function of NDUFS3 (and other pertinent complex I subunits)
will be by means of rationally-designed small molecule probes
specifically targeted to NDUFS3 assembly sites. By this
approach, one could exert a controlled perturbation of complex
I assembly process and monitor its effects on the metabolic
switch phenotype. Currently available crystal structure models
are limited to prokaryotic complex I. In the absence of any
human complex I structure, many groups have modeled the
human subunit based on the available prokaryotic structure with
the caveat that human and prokaryotic complex I vary widely in
subunit composition, number and complexity (Koopman et al.,
2010; Vogel et al., 2007). A more detailed investigation of the
human complex I crystal structure, in future, could lead to a
better understanding of NDUFS3 and other critical complex I
subunits in human health and disease phenotypes.
Finally, our study has clearly demonstrated that the observed
mitochondrial dysfunction is a sufficient condition for the
observation of aerobic glycolysis phenotype but the necessary
condition for sustaining the aerobic glycolysis (or metabolic
switch) in these cell lines is the sustained ROS imbalance as
shown by the assays described in this paper. Our data are in
accordance with a recent study where it was demonstrated that
reactive oxygen species can promote aerobic glycolysis in
chronic leukemia cell lines (Lu et al., 2012). In addition to
establishing the basis for the ROS-mediated aerobic glycolysis,
our study highlights a plausible answer to the original question
namely the maneuverability of metabolic switch phenotype
within the limits of mitochondrial dysfunction. An alternate
way to consolidate these observations will be by development of
a model system with inducible expression of NDUFS3 (and other
critical subunits of mitochondrial complex I) to precisely
Fig. 8. Effects of exogenous antioxidant mimetics on ROS status in the control and NDUFS3-deficient HEK cells. (a,b) Representative ROS mesurements as
assayed by Amplex Red reagent, which reports the net hydrogen peroxide signals. Cells were treated with denoted concentrations of superoxide mimetic MnTBAP
and glutathione precursor, N-acetyl cysteine for 36 hours. Corroborative flow cytometry measurements are shown in c.(d) Average (3 independent experiments)
lactate generation rate measured in the control and NDUFS3-deficient cell lines in the presence of antioxidant mimetic 10 mM MnTBAP to determine the causative
role of ROS status in influencing the observed metabolic switch in the NDUFS3-deficient cells. Lactate values in untreated case were the reference values (100%) for
each cell line.
Mitochondrial NDUFS3 304
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determine the molecular basis of mitochondrial reprogramming
in transformed cells. In conclusion, we believe that we have laid a
novel foundation for studying mitochondrial complex I
dysfunction in transformed cells. Since mitochondrial
metabolism is a fundamental pathway in almost all cell types,
we envision that the ideas described in this paper can be easily
applied to a variety of metabolic disorders that encompass
mitochondrial dysfunction.
We gratefully acknowledge financial support from Susan G. Komen
for the Cure foundation (Career Catalyst Research Award no.
KG090239), National Cancer Institute/National Institutes of Health
(ARRA Stimulus Award no. R21-CA124843), Donna and Jesse
Garber Foundation award and American Cancer Society Inc.
(Research Scholar Award RSG-12-144-01-CCE) (all to V.K.R).
We thank Dr Bruce Gewertz and Dr Leon Fine for their intramural
support and encouragement.
Competing Interests
The authors have no competing interests to declare.
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    • "Since the sensitivity of the AmR assay was rather low in DMEM, we applied Dulbecco's phosphate-buffered saline, in which case excellent assay sensitivity was associated with seriously compromised respiration, both with (up to 50% inhibition) and without AmR (up to 20% inhibition). A brief literature survey indicates that AMR concentrations and media applied are highly variable, ranging from 1 μM AmR in PBS for the study of permeabilized C2C12 myoblasts and myotubes [25], 10 μM AmR in MiR05 for primary human skeletal myotubes [26], to 50 μM AmR in various phosphate-buffered or bicarbonate-buffered saline media investigating N27 cells [27], A549 lung epithelial cells [20], HaCaT keratinocytes [28], or HEK 293T cells [29], or mitochondria prepared from H9c2 rat cardiac myocytes [29] or from HepG2 cells [30]. In the absence of adequate controls it is not clear to which extent these treatments might have affected the results on H2O2 production. "
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    Full-text · Article · Jun 2015
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    • "This suggests that, while overall subunit content of C-I is not different in skeletal muscle of the OGDM women, content of specific, important C-I subunit proteins is reduced, resulting in reduced mitochondrial enzyme activity. Indeed, gene silencing of NDUFS3 and mutations in the NDUFV2 gene have been shown to lead to mitochondrial dysfunction, hypertrophic cardiomyopathy, and Leigh's Syndrome [48,49]. Thus, calcium regulatory control of AMPK phosphorylation and mitochondrial enzyme capacity may have clinical implications that may partially explain why physical activity interventions in women with GDM, for the control glycemia and prevention of its progression to T2DM, are disappointing and often associated with a high non-compliance rate [10,11,50] . "
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