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).

3

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-

9

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.

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(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