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www.sciencemag.org/cgi/content/full/science.1211485/DC1
Supporting Online Material for
Inhibition of Pyruvate Kinase M2 by Reactive Oxygen Species Contributes to Cellular Antioxidant Responses
Dimitrios Anastasiou, George Poulogiannis, John M. Asara, Matthew B. Boxer, Jian-kang Jiang, Min Shen, Gary Bellinger, Atsuo T. Sasaki, Jason W. Locasale,
Douglas S. Auld,* Craig J. Thomas, Matthew G. Vander Heiden, Lewis C. Cantley
*To whom correspondence should be addressed. E-mail:
lewis_cantley@hms.harvard.edu
Published 3 November 2011 on Science Express DOI: 10.1126/science.1211485
This PDF file includes
Materials and Methods Figs. S1 to S10 Full References
1
SUPPORTING ONLINE MATERIAL
MATERIALS AND METHODS
Cell lines, cell culture, virus preparations, PKM2 activator and oxidant treatments
293T and A549 cells were obtained from ATCC and cultured in DMEM (Mediatech)
supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml
streptomycin. H1299 (ATCC) cells were cultured in RPMI (Mediatech) supplemented as above. All
cells were cultured in a humidified incubator at 37 oC/5% CO2 unless otherwise stated. Glucose
concentration in the media was 25 mM (4.5 g/l) unless otherwise stated. Diamide [1,1′-Azobis(N,N-
dimethylformamide), D3648] and H2O2 (H1009) were from Sigma and used as described in the text.
Hypoxia treatments were performed using an InVivo2 400 humidified workstation (Ruskinn, Pencoed,
UK). For all hypoxia treatments (and corresponding normoxic control cultures), the media were
supplemented with 20 mM HEPES buffer. For the experiment in Fig. 1B, A549 cells were washed
once with PBS (37oC), the culture medium was replaced with medium containing 5.6 mM glucose and
the cells were placed for 3 hours under 21% O2 or 1% O2. For experiments where 5.6 mM glucose
was used in any part of the experiment, DMEM without glucose and without sodium pyruvate
(Invitrogen-11966025) or RPMI medium without glucose (Invitrogen-1187920) were used, and
supplemented with antibiotics as above, 10% dialyzed FCS (Invitrogen-26400044) and D-(+)-glucose
(Sigma-G7021) at the indicated final concentrations.
Cells expressing specific Flag-tagged isoforms of mouse pyruvate kinase M, or mutants
thereof, in the absence of endogenous PKM2 (in text referred to as “Flag-PKMxxx/kd” cells) were
derived by first infecting cells with retroviruses to express the relevant cDNA, followed by shRNA-
mediated knock-down of endogenous PKM2 with a lentivirus-expressed shRNA (S1). Retroviruses
were produced in 293T cells by co-transfection of a plasmid expressing the amphotropic receptor
gene and pLHCX-based vectors expressing the cDNA of interest fused to the C-terminus of a
sequence encoding the Flag-epitope. Point mutations were introduced by two-step PCR. Viral
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supernatants were harvested at 48 h post-transfection, supplemented with 4 µg/ml polybrene and
applied to target cells for 6-8 hours before replacing with normal growth media. Infection was repeated
with fresh viral supernatants the following day after which cells were allowed to recover in normal
medium for 12-16 hours prior to selection with hygromycin (300 µg/ml) for at least 10 days.
Lentiviruses were produced in 293T cells by co-transfection of plasmids expressing gag/pol,
rev and vsvg with a pLKO vector encoding a short hairpin targeting human PKM2 (S1). Selection was
achieved with puromycin (2 µg/ml) for at least 4 days.
PKM2 activators were described in (S2). DASA-10 at 10 µM was used in all experiments
unless otherwise stated.
Cell harvesting, lysis, SDS-PAGE and western blotting
Cells attached to culture dishes were quickly washed once with a large volume (20-30 ml) of
ice-cold PBS, snap-frozen in a liquid nitrogen bath and stored at -80oC until further processing. Cells
were lysed in PK lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Igepal-630)
supplemented freshly prior to usage with protease inhibitors [10 μg/ml phenymethylsulfonyl fluoride, 4
μg/ml aprotinin, 4 μg/ml leupeptin, and 4 μg/ml pepstatin (pH 7.4)] and 1 mM DTT where applicable.
For detection of oxidized PKM2 by SDS-PAGE, cells were lysed in de-gassed lysis buffer without
reducing agents until electrophoresis within 1 h post-lysis. PK activity assays from total lysates of
normally growing cells indicate that within this time frame (<1hour), PKM2 activity is not significantly
affected by exposure to ambient oxygen concentrations based on the fact that DTT does not enhance
PKM2 activity from untreated (no oxidants) cells. For reducing SDS-PAGE, lysates were mixed with
SDS-PAGE loading buffer (50 mM Tris-HCl pH 8.8, 1% w/v SDS, 2.5% glycerol, 0.001% w/v
bromophenol blue and 143 mM β-mercaptoethanol, final conc.) and boiled for 10’. For non-reducing
SDS-PAGE, β-mercaptoethanol was omitted from the gel loading buffer and samples were not boiled.
Antibodies for western blotting were: PKM1/2 (goat, 1:2000, Abcam-cat. # ab6191-5), PKM2 (rabbit,
1:1000, Cell Signaling Technology-cat. # 4053) and Flag (mouse, 1:5000, Sigma-cat. # F1804).
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Immunoprecipitation
Cells were harvested as above and lysed in 700 µl PK lysis buffer supplemented with protease
inhibitors and 1 mM DTT, where applicable. Lysates were centrifuged (20,000xg, 10’, at 4 oC),
supernatants were transferred to fresh eppendorf tubes containing 20 µl of 50% Flag-agarose (Sigma-
A2220) bead slurry in PK lysis buffer and incubated rotating at 4 oC for 1 hr. Under these conditions,
lysates were immunodepleted of detectable Flag-tagged proteins. Immunoprecipitates were washed 4
times with PK lysis buffer (1ml=100 bead-volumes per wash) then eluted from beads with 3xFlag
peptide (150 µg/ml final concentration, Sigma, F4799, dissolved in 50 mM Tris-HCl pH 7.4, 150 mM
NaCl) for 30’ rotating at 4oC. Following a brief centrifugation of the beads, eluates were transferred to
fresh eppendorf tubes, supplemented with SDS-PAGE loading buffer and analyzed by SDS-PAGE.
Biotin labeling of oxidized PKM2
Cells were lysed for 15 min. on ice in biotin labeling lysis buffer (BLLB: 50 mM Tris-HCl pH
7.0, 5 mM EDTA, 120 mM NaCl, 0.5% Igepal-630) containing protease inhibitors (as above) and 100
mM maleimide (Sigma-129585). Insoluble material was then removed by centrifugation at 20,000xg
for 10 min. at 4oC, the cleared supernatant was transferred to a fresh eppendorf tube and protein
concentration was determined by the Bradford assay. Protein concentration was adjusted to 1 µg/µl
with BLLB, SDS was added from a 10% stock to a final concentration of 1% and the cell lysates were
incubated at room temperature for 2 hours rotating. To remove unreacted maleimide, proteins were
subsequently precipitated by adding 5 volumes of acetone pre-equilibrated at -20oC and incubated for
20 min. at -20oC. The preparations were centrifuged at 20,000xg for 10 min. at 4 oC, supernatants
removed and discarded and precipitated protein pellet was air-dried. The pellet was then resuspended
in 200 µl BLLB containing 1% SDS, 10 mM DTT and 0.1 mM biotin-maleimide (Sigma-B1267, stock
dissolved in dimethylformamide) to reduce the remaining, previously oxidized, sulfhydryl groups and
allow their reaction with biotin-maleimide. Proteins were again precipitated with 5 volumes of
methanol (-20oC) as above, the dried pellet was resuspended in 500 µl of BLLB, incubated with 10 µl
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of a 50% slurry of streptavidin-sepharose beads (GE Healthcare-17511301) rotating at 4 oC for 2
hours. The beads were then washed 4 times with BLLB and resuspended in SDS-PAGE loading
buffer for SDS-PAGE analysis and western blotting with the indicated antibodies.
Pyruvate kinase activity assays
PK activity was measured by monitoring pyruvate-dependent conversion of NADH to NAD+ by
lactate dehydrogenase (LDH) (S1). Cells were lysed as above and protein concentration was
determined by the Bradford assay. Immediately prior to start of the assay, 1 µg of total protein was
mixed with 1x pyruvate kinase reaction buffer [50 mM Tris-HCl pH 7.5, 100 mM KCl and 5 mM MgCl2
containing 0.5 mM PEP (Sigma-P0564), 0.6 mM ADP (Sigma-A5285), 180 µM NADH (Sigma-N8129),
0.015% Brij, 8 units LDH (Sigma-L1254), 1mM DTT (where applicable), and 200 µM FBP (Fluka-
47810)(where applicable)]. The final reaction volume was 100 µl in 96-well plates. For the experiment
in fig. S3B, recombinant PKM2 was produced in E. coli and purified as in (S3) and after treatments as
indicated in the figure legend, catalase (Sigma-C1345, 1 mg/ml stock in 50 mM KPO4 at pH 7.0) was
used at 10 µg/ml. For the experiment in fig. S9D, the amino acid sequences of the peptides were:
GGAVDDDYAQFANGG (M2tide) and GGAVDDDpYAQFANGG (P-M2tide) (S3).
Metabolite analysis by targeted liquid-chromatography tandem mass spectrometry (LC-MS/MS)
48 hours prior to each experiment, 2.5x105 cells were seeded in 6 cm dishes in media without
sodium pyruvate, containing 10% dialyzed FCS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml
streptomycin. Media were changed at t=-24 h and t=-2 h. At t=0h diamide was added directly to the
media, where applicable, at a final concentration of 250 µM and cells were harvested at the indicated
time points as follows: media were aspirated and metabolites were extracted with 1.5 ml of 4:1 v/v
MeOH/H2O equilibrated at -80 oC. The extract and cells were scraped and collected into 15 ml conical
tubes and centrifuged for 5 min. at 690xg and solvent in the resulting supernatant was evaporated
using a speed-vac. Samples were re-suspended in 20L HPLC-grade water for mass spectrometry. 8
5
μL were injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/Sciex)
coupled to a Prominence UFLC system (Shimadzu) via selected reaction monitoring (SRM) of 249, in
total, endogenous water-soluble metabolites. Some metabolites were targeted in both positive and
negative ion mode for a total of 298 SRM transitions. ESI voltage was +4900V in positive ion mode
and –4500V in negative ion mode. The dwell time was 5 ms per SRM transition and the total cycle
time was 2.09 seconds. Approximately 8-10 data points were acquired per detected metabolite.
Samples were delivered to the MS via normal phase chromatography using a 2.0 mm i.d. x 15 cm
Luna NH2 HILIC column (Phenomenex) at 285 μL/min. Gradients were run starting from 85% buffer B
(HPLC grade acetonitrile) to 42% B from 0-5 minutes; 42% B to 0% B from 5-16 minutes; 0% B was
held from 16-24 minutes; 0% B to 85% B from 24-25 minutes; 85% B was held for 7 minutes to re-
equilibrate the column. Buffer A was comprised of 20 mM ammonium hydroxide/20 mM ammonium
acetate (pH=9.0) in 95:5 water:acetonitrile. Peak areas from the total ion current for each metabolite
SRM transition were integrated using MultiQuant v1.1 software (AB/Sciex).
Pentose phosphate pathway (PPP)-dependent glucose oxidation to CO2
PPP activity was measured using an adaptation of previously published procedures (S4, 5).
More specifically, 4,500 cells were seeded in 96-well plates 24 hours prior to the experiment. Media
were supplemented with 5 µCi/ml of [1-14C]-glucose (specific activity 45-60 mCi/mmol) or [6-14C]-
glucose (specific activity 50-62 mCi/mmol) and treatment compounds (diamide and PKM2 activator)
as indicated in the text and figures, in a final volume of 100 µl. The wells were overlaid with 3mm
Whatman paper which had been impregnated just prior to use in a saturated Ba(OH)2 solution
(prepared with boiled water) and blotted dry. Released 14CO2 was captured immediately above each
well by forming insoluble Ba14CO3 on the filter. The plate lid was placed on top of the filter; the plate
was sealed with parafilm and was incubated at 37%/5% CO2 for 3 hours. The Whatman paper was
then removed, placed in an acetone bath, air-dried and incubated at 110 oC for 5 minutes. The filter
was then cut into pieces each corresponding to a well of the plate, placed in a scintillation vial
6
containing scintillation fluid and radioactivity was measured in a Beckman LS6000SC scintillation
counter. The release of 14CO2 from [1-14C]-glucose, provides a quantitative measure of flux through
the PPP enzyme, 6-phosphogluconate dehydrogenase, while 14CO2 release from [6-14C]-glucose in a
parallel experiment provides a quantification of TCA cycle-dependent CO2 production from glucose.
PPP-dependent CO2 production was calculated as the difference between 14CO2 derived from [1-14C]-
glucose and 14CO2 derived from [6-14C]-glucose.
ROS and GSH measurements
For ROS measurements, the medium was aspirated, cells were washed 1x with PBS and
incubated with PBS containing 1 µM chloromethyl-H2DCFDA (CM-H2DCFDA, Invitrogen-C6827) in
DMSO for 30’ at 37 oC/5% CO2. The dye was then removed and media containing H2O2 were added
at the indicated concentrations and times. For ROS measurements under hypoxia (fig. S1), cells were
washed 1x with warm PBS and incubated for 2 hours under 1% O2 in media containing 5.6 mM
glucose. The media were then removed and retained, and cells were loaded with 1 µM CM-H2DCFDA
in PBS for 30 min. at which point the PBS was removed and the same media were replaced on the
cells. All buffers and media used following the replacement of the media upon starting the hypoxic
treatment had been pre-equilibrated under the same hypoxic conditions at least overnight. Following
these procedures, cells were trypsinized, centrifuged, resuspended in 500 µl PBS and maintained on
ice, in the dark, until analysis by flow cytometry (FACScan, BD Biosciences).
For GSH measurements, the medium was aspirated, cells were washed 1x with PBS and
incubated with PBS containing 12.5 µM ThiolTrackerTM Violet (Molecular Probes-T10095) for 30’, at
37 oC/5% CO2. ThiolTrackerTM Violet conjugates to reduced (GSH) but not to oxidized glutathione,
therefore, ThiolTrackerTM Violet fluorescence corresponds to intracellular GSH concentration. Cells
were subsequently harvested by trypsinization and processed for flow cytometry as above.
7
BrdU incorporation
Cells were seeded 48 hours prior to BrdU labeling. At t=-24h, media were refreshed and at
t=0h BrdU was added directly into the media at a final concentration of 10 µM for 30’. Cells were then
trypsinized, resuspended in the same culture media, centrifuged (300xg, 5’ at room temperature),
homogeneously resuspended in 300 µl of ice-cold PBS and immediately transferred to ice. 700 µl of
100% ethanol pre-equilibrated at -20 oC were added drop-wise to each cell suspension, and fixed
cells were stored at -20 oC until further processing within 7 days. Fixed cells were washed with
PBS/0.5% BSA, resuspended in 2N HCl for 20’ to denature DNA and expose incorporated BrdU,
washed with PBS/0.5% BSA, neutralized in 100 mM sodium citrate pH 7.5 and stained with anti-BrdU
antibody (clone 3D4, BD Pharmingen, 51-33284X and corresponding isotype control) in
permeabilization buffer (PBS/0.5% BSA containing 0.5% Tween-20) for 30’ at room temperature.
Cells were then washed with PBS/0.5% BSA, centrifuged, resuspended in FACS solution (38 mM Na-
Citrate pH 7.5, 69 µM propidium iodide, freshly supplemented with 20 µg/ml RNase) incubated for 20’
at 37oC and analyzed by flow cytometry (FACScan, BD Biosciences).
Cell mass accumulation assay
20,000 cells were seeded in 12-well plates at day -1 in glucose-free media supplemented with
25 mM or 5.6 mM D-glucose and incubated at the indicated O2 concentrations. In all cases media
contained 10% dialyzed FCS and 20 mM HEPES pH 8.0. At day 0 the media were replaced with fresh
media that had been equilibrated since the time of cell seeding at 37oC under the corresponding
oxygen concentrations and supplemented with 4 mM glutathione monoethyl ester (GSH-MEE,
Calbiochem-353905), a cell-permeable GSH analogue, as indicated. Cells were fixed at days 0, 2, 4
and 7 as follows: cells attached on the plate were washed once with PBS at room temperature,
incubated for 10’ in PBS-buffered 10% formalin, washed twice with PBS and stored at 4oC until
completion of the experiment and at least overnight. Cells were then washed with ice-cold PBS,
stained with 0.1% w/v crystal violet in 20% methanol, shaking for 15’ at room temperature, and
8
washed with water twice for 10’ each; the plates were then dried on air. Cell-bound crystal violet was
solubilized in 1 ml 10% v/v acetic acid and, because the amount of dye bound to cells is proportional
to the number of cells, accumulation of cell mass was assessed by measurement of crystal violet
absorbance at 595 nm in a spectrophotometer.
MTS cell viability assay
2,000 cells were seeded in 96-well plates 24 h prior to treatment start. CellTiter96® AQueous
(Promega-G5421) was used according to the manufacturer’s protocol to assess cell viability following
oxidant and PKM2 activator combination treatments. MTS: (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).
Evaluation of cell death by propidium iodide incorporation
Cells were seeded in 6-well plates 24-48 hours prior to the experiment. Following treatment
with oxidants, cells were trypsinized, centrifuged, re-suspended in 500 µl FACS solution [38 mM Na-
Citrate pH 7.5, 69 µM propidium iodide (PI)] and analyzed by flow cytometry as described above. For
this assay, H1299 cells were used as they exhibited faster death kinetics (up to 100% death after 3
hours of treatment with maximal diamide concentration) compared to A549 cells (<80% after 24h
under the same conditions). In the context of PKM2 activator treatment, the rapid induction of
diamide-induced death in H1299 cells allowed us to assay cellular viability following relatively short
treatment times (3 hours), thereby eliminating the possibility of potential secondary transcriptional or
metabolic effects of the activator.
Xenograft assay
H1299 cells were engineered to express Flag-PKM2 or Flag-PKM2(C358S) in the absence of
endogenous PKM2 as described above. 5x106 cells resuspended in 100 µl culture medium were
mixed with an equal volume of MatrigelTM (basement membrane-high concentration, BD Biosciences-
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354248) and injected subcutaneously in the left (wild-type PKM2) or right (mutant C358S PKM2) flank
of each of 10 nu/nu mice (Charles River-strain 088). The mice were then randomly divided into two
groups, one given access to standard water and the other to water supplemented with 40 mM NAC for
the entire duration of the experiment. Tumor size was monitored by measuring tumor width and length
every 3-4 days. At the end-point (day 36 post-injection), tumors were excised, measured, weighted
and photographed; small tissue samples were dissected away, frozen in liquid nitrogen and stored at
-80oC until further processing for western blotting. Water (as described above) and food were
available to the mice ad lib; animal housing and handling was in accordance to IACUC regulations.
Statistical analysis
For metabolomics analyses, prior to any statistical computation, the metabolite measurement
data were log2-transformed and normalised using the quantile approach implemented in the limma
package in R 2.12, which ensures that the intensities of all metabolite measurements have the same
empirical distribution across different sample runs. The empirical Bayes (eBayes) shrinkage of the
standard errors towards a common value approach (S6) was used to identify the metabolites whose
levels were significantly different between DASA-10 and control (DMSO) treatments at each of the
respective time points (Fig. 3B). For comparison of tumor weights and volumes in the xenograft
experiment, one-way ANOVA was used followed by Tukey’s multiple comparison test, in GraphPad
Prism® 5.03. For all other statistical analyses, two-way ANOVA (GraphPad Prism® 5.03) or unpaired
Student’s t-test (Excel) were used as indicated in the respective figure legends.
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Supplementary figures S1-S10 – LEGENDS
Figure S1. Diamide, H2O2 and 1% O2 elicit comparable increases in intracellular ROS
concentrations.
H1299 cells were loaded with CM-H2DCFDA which exhibits an increase in fluorescence upon
oxidation. Cells were then treated for 20 min. with the indicated doses of diamide or H2O2 in standard
growth media. For the measurement of hypoxia-induced ROS, cells were incubated for 2 hours under
1% O2 in medium containing 5.6 mM glucose. Under the same O2 atmosphere, cells were then loaded
with CM-H2DCFDA in PBS (pre-equilibrated at 1% O2) and after 30 min. the original treatment
medium was replaced and cells were incubated for another 30 min. (total time under 1% O2 =3 hours).
Following all treatments, cells were harvested by trypsinization and ROS-dependent fluorescence was
measured by flow cytometry.
Figure S2. Structure of the PKM2 tetramer and identification of Cys358 as the mediator of the
oxidant-induced electrophoretic mobility shift.
(A) Structure of the PKM2 tetramer bound to FBP (PDB ID: 1T5A) (S7). Cys31 and Cys424 are shown
in magenta. Structure image [also in (B)] was generated in PyMOL (DeLano Scientific).
(B) Highlight of the β-barrel structure and catalytic site at the core of the PKM2 monomer. Residues
involved in catalysis are represented as sticks; molecules shown in ball representations are: oxalate,
a mimic of the reaction intermediate enolpyruvate (S8), Mg2+ and K+ ions required for catalysis, free
phosphate (P) and Cys358.
(C) A549 cells engineered to express Flag-PKM2 or the indicated mutants, were treated with 250 µM
diamide for 15 min., lysed and analyzed by reducing (bottom panel) or non-reducing (top panel) SDS-
PAGE. The data in the upper panel of Fig. 2B were cropped from this picture. K433E corresponds to a
PKM2 mutant that cannot bind phosphotyrosine-containing peptides (S3).
11
Figure S3. Structures of PKM2 activators used in this study, dose-dependent activation of
PKM2 by DASA-10 and impaired ability of PKM2 activator DASA-10 to fully activate PKM2
when added to diamide-treated cells after lysis.
(A) Chemical structures of DASA-10 {NCGC00181061, 1-(2,6-difluorophenylsulfonyl)-4-(2,3-
dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine} and DASA-58 {NCGC00185916, ML203, 3-(4-(2,3-
dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-1,4-diazepan-1-ylsulfonyl)aniline}.
(B) PKM2 expressed in bacteria was purified under reducing conditions and the reducing agent was
then removed by gel filtration. Then, PKM2 (~2 µg/µl) was treated with DMSO or DASA-10 (1 µM) and
the indicated amounts of H2O2 for 30 minutes at room temperature. The preparations were then
diluted 100-fold in PK assay buffer containing catalase and PK activity was assayed.
(C) A549 cells were treated with 0, 1, 10, 25 or 50 µM DASA-10 for 1 hour, lysed and assayed for
PKM2 activity. Note that maximal PKM2 activation was achieved at 50 µM DASA-10 concentration.
(D) Following a first round of PKM2 activity assays (presented in Fig. 2E), the same lysates of cells
treated or not with diamide in the absence of activator from Fig. 2E were subsequently supplemented
with DASA-10 (final concentration=50 µM), incubated on ice for 1h and pyruvate kinase activity was
assayed as in Fig. 2E.
Figure S4. Selected PKM2 activator concentration restores diamide-induced PKM2 inhibition to
levels found in untreated cells.
A549 cells were treated with 10 µM PKM2 activator DASA-10 at t=-1h and diamide (250 µM final
concentration) was added directly to the media at t=-15’. Cells were harvested at t=0 and PKM2
activity was assayed. Note that, at this concentration, DASA-10 rescues PKM2 activity to levels found
in cells not treated with diamide (arrows). Thus, under these conditions which were also utilized for
the analysis of metabolic changes in Fig. 3B, the PKM2 activator is more likely to reveal effects in
metabolism due to the prevention of diamide-induced activity inhibition rather than due to hyper-
activation of PKM2.
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Figure S5. Effects of PKM2 activator on cellular GSH and H2O2-induced ROS levels.
(A) GSH measurements. Experiments were performed exactly as in Fig. 3C (right), but PKM2
activator DASA-58 (S2) instead of DASA-10 was used.
(B) A549 Flag-PKM1/kd or Flag-PKM2/kd cells (generated as in Fig. 1D) were treated with 10 µM
DASA-10 and metabolites were extracted at the indicated times for mass spectrometry-based
quantification. The ordinate represents ion counts (N=1).
(C) Measurement of H2O2-induced intracellular ROS levels. Experiments were performed exactly as in
Fig. 3D, but PKM2 activator DASA-58 instead of DASA-10 was used. Representative data from N=2
experiments are shown.
Figure S6. Deletion of Pkm exon 10 in Pkm2flox/flox MEFs leads to equivalent levels of PKM1
expression and impairs detoxification of H2O2-induced intracellular ROS.
(A) Schematic representation of Pkm gene intron-exon organization. Exon 9 (green box) encodes the
PKM1-specific region and exon 10 (red box) encodes the PKM2-specific region of the protein.
(B) Upper scheme describes the infection protocol with tamoxifen-inducible Cre recombinase and the
Cre induction strategy with 4-hydroxytamoxifen (4-HT) to delete exon 10 in Pkm2flox/flox MEFs. Middle
panel illustrates time-dependent abrogation of PKM2 protein expression following Cre-induction and
concomitant emergence of PKM1 protein expression. Note that total PK levels detected by western
blot (with an antibody recognizing both PKM1 and PKM2) are unchanged. Lower panel: PK activity
assays from the same lysates indicate that, following Cre induction, the detectable PK activity is not
inducible by FBP consistent with the western blot data that show a switch of expression from FBP-
sensitive PKM2 to FBP-insensitive PKM1. In addition, note that, as with the protein levels, total
cellular PK activity is also comparable to PKM2 (0 nM 4-HT, +FBP) in the presence of physiological
concentrations of FBP (200 µM) throughout the experiment. Thus, any effects observed in functional
assays [shown in (C)] are because of differential regulation of PKM1 compared to PKM2 rather than
aberrant overexpression of PKM1.
13
(C) E12.5 MEFs from Pkm2flox/flox mice were infected with retrovirus expressing tamoxifen-inducible
Cre-recombinase as described in (B). Cre activity was induced with 600 nM 4-hydroxytamoxifen (+4-
HT) at day 0 while a separate cell population was cultured in parallel without 4-HT treatment (- 4-HT),
as in (B). At day 6, cells were treated with 5 mM H2O2 for 10 min. and ROS levels were measured as
in Fig. 3D (p value calculated by 2-way ANOVA with Bonferroni post-test, N=3).
Figure S7. Effects of PKM2 activator on cellular viability under oxidative stress.
A549 cells were seeded in 96-well plates and treated with increasing doses of H2O2 (A) or diamide (B)
in the presence of 10 µM PKM2 activator DASA-10 or DMSO. Cell viability was assayed 24-36 hours
after treatment start using an MTS-based colorimetric assay (see Materials and Methods).
Figure S8. Proliferation profiles of Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells in various
glucose and oxygen concentrations.
(A) Cell mass accumulation of H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells under 21% O2
in media containing 25 mM glucose was measured as in Fig. 4C.
(B) Cell cycle distribution profiles of H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells [cultured
in 25 mM glucose/21% O2 as in (A)] derived from flow cytometry analysis of BrdU-labelled cells (N=1).
(C) Cell mass accumulation of H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells under 1% O2 in
media containing 25 mM glucose was measured as in Fig. 4C. Note that although no difference in cell
mass accumulation was evident up to the day 4 time point, at day 7 there was a decrease in cell
numbers which was more pronounced when the PKM2(C358S) mutant was expressed.
(D) Cell mass accumulation of H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells under 21% O2
in media containing 5.6 mM glucose was measured as in Fig. 4C. (p=0.0041, 2-way ANOVA, N=3)
14
Figure S9. Cys358 mediates inhibition of PKM2 by hypoxia-induced ROS; Cys358 mutation to
Ser358 does not affect PKM2 inhibition by phosphotyrosine peptide binding.
(A) Upper panel: H1299 cells were incubated for 3 hours under 21% O2 or 1% O2 in media containing
5.6 mM glucose in the presence or absence of 2 mM NAC. Cells were then lysed under non-reducing
conditions and oxidized proteins were labeled using the biotin-switch method as in Fig. 2B. Lower
panel: H1299 cells were treated as above, lysed under non-reducing conditions and analyzed directly
by SDS-PAGE under reducing or non-reducing conditions.
(B) H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells were incubated for 3 hours under 21% O2
(“hypoxia: -”) or 1% O2 (“hypoxia: +”) in media containing 5.6 mM glucose. Cells were then lysed
under non-reducing (-DTT) or reducing (+DTT) conditions and oxidized proteins were labeled using
the biotin-switch method.
(C) H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells were treated as in (B) in the presence or
absence of 2 mM NAC, lysed under non-reducing conditions and assayed for PKM2 activity.
(D) H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells were lysed and incubated for 10 min. on
ice with 50 µM of either a phosphotyrosine-containing peptide with an amino acid sequence that
corresponds to the optimal motif for PKM2 binding (P-M2tide) or a control non-phosphorylated peptide
with the same amino acid sequence (M2tide)(S3). After lysis, PKM2 activity was assayed.
Figure S10. Effect of PKM2(C358S) mutant expression on the tumorigenic potential of H1299
cells in mouse xenograft assays.
(A) Overview of the xenograft experiment setup (upper panel) and photographs of the mice (lower
panel) bearing the tumors depicted in Fig. 4E just prior to dissection.
(B) Western blots of lysates from the H1299 Flag-PKM2/kd and Flag-PKM2(C358S)/kd cells used in
the xenograft experiment (“injected cells”) and corresponding parental cells prior to endogenous
PKM2 knock-down analyzed alongside lysates from the derived tumors (“Xenografts”). denotes
Flag-PKM2(WT) or Flag-PKM2(C358S) bands; denotes endogenous PKM2 bands. Note that at the
15
end of the experiment, all tumors had retained expression of the Flag-tagged PKM2 in amounts
comparable to those in the injected cells and endogenous PKM2 expression remained low; therefore
differences in final tumor size could be attributed to the specific PKM2 isoform expressed. Numbers
throughout the figure correspond to tumors in Fig. 4E.
References and Notes 1. N. K. Tonks, Cell 121, 667 (2005). 2. K. E. Wellen, C. B. Thompson, Mol Cell 40, 323 (2010). 3. O. Vafa et al., Mol Cell 9, 1031 (2002). 4. V. Nogueira et al., Cancer Cell 14, 458 (2008). 5. A. A. Sablina et al., Nat Med 11, 1306 (2005). 6. K. Bensaad, E. C. Cheung, K. H. Vousden, EMBO J 28, 3015 (2009). 7. W. Hu et al., Proc Natl Acad Sci U S A 107, 7455 (2010). 8. S. Reuter, S. C. Gupta, M. M. Chaturvedi, B. B. Aggarwal, Free Radic Biol Med 49, 1603
(2010). 9. B. Halliwell, Biochem J 401, 1 (2007). 10. Z. T. Schafer et al., Nature 461, 109 (2009). 11.K. Ishikawa et al., Science 320, 661 (2008). 12. F. Weinberg et al., Proc Natl Acad Sci U S A 107, 8788 (2010). 13. A. J. Levine, A. M. Puzio-Kuter, Science 330, 1340 (2010). 14. P. P. Pandolfi et al., EMBO J 14, 5209 (1995). 15. S. Filosa et al., Biochem J 370, 935 (2003). 16. M. G. Vander Heiden, L. C. Cantley, C. B. Thompson, Science 324, 1029 (2009). 17. H. R. Christofk et al., Nature 452, 230 (2008). 18. H. R. Christofk, M. G. Vander Heiden, N. Wu, J. M. Asara, L. C. Cantley, Nature 452, 181
(2008). 19. P. Maeba, B. D. Sanwal, J Biol Chem 243, 448 (1968). 20. B. McDonagh, S. Ogueta, G. Lasarte, C. A. Padilla, J. A. Barcena, J Proteomics 72, 677
(2009). 21. D. A. Butterfield, R. Sultana, J Alzheimers Dis 12, 61 (2007). 22. R. C. Cumming et al., J Biol Chem 279, 21749 (2004). 23. J. K. Brunelle et al., Cell Metab 1, 409 (2005). 24. Materials and methods are available as supporting material on Science Online. 25. E. Eigenbrodt, M. Reinacher, U. Scheefers-Borchel, H. Scheefers, R. Friis, Crit Rev Oncog 3,
91 (1992). 26. Y. M. Janssen-Heininger et al., Free Radic Biol Med 45, 1 (2008). 27. J. D. Dombrauckas, B. D. Santarsiero, A. D. Mesecar, Biochemistry 44, 9417 (2005). 28. M. B. Boxer et al., J Med Chem 53, 1048 (2010). 29. C. Le Goffe et al., Biochem J 364, 349 (2002).
Supporting References S1. H. R. Christofk et al., Nature 452, 230 (2008). S2. M. B. Boxer et al., J Med Chem 53, 1048 (2010). S3. H. R. Christofk, M. G. Vander Heiden, N. Wu, J. M. Asara, L. C. Cantley, Nature 452, 181
(2008). S4. S. W. Tuttle et al., J Biol Chem 282, 36790 (2007). S5. H. Tabor, C. W. Tabor, E. W. Hafner, J Bacteriol 128, 485 (1976). S6. G. K. Smyth, Stat Appl Genet Mol Biol 3(1), Article3 (2004). S7. J. D. Dombrauckas, B. D. Santarsiero, A. D. Mesecar, Biochemistry 44, 9417 (2005). S8. G. Michaels, Y. Milner, G. H. Reed, Biochemistry 14, 3213 (1975).
0
1
2
3
4
5
6
7
8
9
10
- H 125 250 0.1 1 5
Intr
ace
llula
r R
OS
co
nce
ntr
atio
n
(re
lative
to
un
tre
ate
d)
Diamide
(µM)
H2O2
(mM)
1% O2
H = Hypoxia
Supplementary figure S1
Anastasiou et al.
Supplementary figure S2
Anastasiou et al.
180o
C
A
Diamide:
Flag-PKM2
WT K433E C358S C31S C424L
Non-reducing
Reducing
WB: Flag
B
0
50
100
150
200
250
300
P
KM
2 a
ctivity (
%)
- DTT
+ DTT
Supplementary figure S3
Anastasiou et al.
DASA-10
(NCGC00181061)
DASA-58
(NCGC00185916)
C
A
N N S
O
S
O
O
O
O
NH2
O
N N S
O
O
S
O
O
F
F O
O
D
Diamide Diamide
DASA-10:
(post-lysis)
0
20
40
60
80
100
120
0 1 10 50
PK
M2
activity (
%)
DMSO
0
20
40
60
80
100
120
0 1 10 50
PK
M2
activity (
%)
DASA-10
H2O2 (µM) H2O2 (µM)
B
0
20
40
60
80
100
120
140
160
180
200
PK
M2
activity (
%)
Supplementary figure S4
Anastasiou et al.
Diamide:
DASA-10:
B A
Supplementary figure S5
Anastasiou et al.
C
0
20
40
60
80
100
120
DMSO DASA-58
GS
H c
on
ce
ntr
ation
0
5
10
15
20
25
30
35
0 1 5 10
Intr
ace
llula
r R
OS
co
nce
ntr
ation
[H2O2] mM
DMSO
DASA-58
p=0.0076
0
2
4
6
8
10
12
14
16
- 4-HT + 4-HT
Intr
ace
llula
r R
OS
co
nce
ntr
ation
-H₂O₂
+H₂O₂
Day 4
0
50
100
150
200
250
0 600 900 0 600 900 0 600 900
DAY 2 DAY 4 DAY 6
PK
activity (
%)
-FBP
+FBP
WB: PKM1
WB: PKM2
WB: PKM1/2
Day 2 Day 6
4-HT (nM): - - -
harvest time:
+ 4-HT + 4-HT Std. growth medium
Infect
& select
(puromycin)
4-HT (nM): Supplementary figure S6
Anastasiou et al.
B A
C ROS (MEFs)
Day 0
p<0.001
Supplementary figure S7
Anastasiou et al.
A B
0
10
20
30
40
50
60
WT C358S
% o
f ce
ll p
op
ula
tio
n
G1
S
G2/M
Supplementary figure S8
Anastasiou et al.
A B
Flag-PKM2(X)/kd
C
X=
25 mM glucose
21% O2
25 mM glucose
1% O2
0
10
20
30
40
0 2 4 7
Re
lative
ce
ll m
ass
Time (days)
PKM2(WT)
PKM2(C358S)
0
2
4
6
8
10
0 2 4 7
Rela
tive
ce
ll m
ass
Time (days)
PKM2(WT)
PKM2(C358S)
D
0
5
10
15
20
25
0 2 4 7
Rela
tive
ce
ll m
ass
Time (days)
PKM2(WT)
PKM2(C358S)
5.6 mM glucose
21% O2
40
50
60
70
80
90
100
110
120
- + - +
21% O2 1% O2
PK
M2
activity (
%)
PKM2(WT)
PKM2(C358S) p=0.025
NAC:
Supplementary figure S9
Anastasiou et al.
C
0
20
40
60
80
100
120
M2
tide
P-M
2tide
M2
tide
P-M
2tide
PKM2(WT) PKM2(C358S)
PK
M2
activity (
%)
D
1% O2 21% O2
Non-reducing
Reducing
NAC:
WB
: PK
M2
Biotin pull-down
WCE
A
B WT C358S
Hypoxia:
Flag-PKM2:
DTT:
WB
: Fla
g
Biotin pull-down
WCE
: Reduced PKM2 : Oxidized PKM2
A
WT C358S
72
56
72
72
56
Xenografts Cell lines
PKM2 shRNA:
Flag-PKM2:
WT
(left flank)
C358S
(right flank)
- NAC
+ NAC
WT
C35
8S
WT
C35
8S
Flag
72
56 PKM2
Flag
PKM2
B
1 2 3 4 5
6 7 8 9 10
Std
. w
ate
r
56 1 2 3 4 5 1 2 3 4 5
6 7 8 9 10 6 7 8 9 10
injected
cells
WB:
40 m
M N
AC
Supplementary figure S10
Anastasiou et al.
: Flag-PKM2 or Flag-PKM2(C358S)
: endogenous PKM2
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