Biochem. J. (2012) 448, 165–169 (Printed in Great Britain) doi:10.1042/BJ20121225 165

ACCELERATED PUBLICATIONProtein kinase Cβ is critical for the metabolic switch to glycolysisfollowing B-cell antigen receptor engagementDerek BLAIR1, Fay J. DUFORT and Thomas C. CHILES2

Department of Biology, Boston College, 140 Commonwealth Avenue, 414 Higgins Hall, Chestnut Hill, MA 02467, U.S.A.

Signals derived from the BCR (B-cell antigen receptor) controlsurvival, development and antigenic responses. One mechanismby which BCR signals may mediate these responses is byregulating cell metabolism. Indeed, the bioenergetic demandsof naıve B-cells increase following BCR engagement and arecharacterized by a metabolic switch to aerobic glycolysis;however, the signalling pathways involved in this metabolicreprogramming are poorly defined. The PKC (protein kinaseC) family plays an integral role in B-cell survival and antigenicresponses. Using pharmacological inhibition and mice deficient inPKCβ, we demonstrate an essential role of PKCβ in BCR-inducedglycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit

glycolytic rates comparable with those of wild-type B-cellsfollowing BCR cross-linking. The induction of several glycolyticgenes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results indecreased survival of B-cells despite BCR engagement. Theresults establish a definitive role for PKCβ in the metabolic switchto glycolysis following BCR engagement of naıve B-cells.

Key words: B-cell antigen receptor (BCR), B-cell, cell survival,glycolysis, metabolism, protein kinase C (PKC).

INTRODUCTION

B-cell homoeostasis is critical for maintaining adaptive immunityand to avoid autoimmune and immunodeficiency diseases. Inkeeping with this, B-cells are dependent upon extrinsic signalsto maintain survival during development in the bone marrowand the periphery and for cellular homoeostasis. Responsesof B-cells to antigens are associated with the activation ofPKC (protein kinase C), which comprise a subfamily of 11closely related serine/threonine kinase isoforms [1]. A keychallenge is to identify PKC isoform-specific substrates and toelucidate their physiological significance in the context of B-cell antigenic responses. Various genetic and biochemical studiessuggest distinct biological functions for several PKC isoformsin B-cells [1–3]. PKCδ plays a role in the regulation of B-cell responses to self-antigens [2]. In contrast, mice deficientin PKCβ (PKCβ − / − ) exhibit impaired TI-2 (T-independenttype 2) responses and poor survival in the absence of stimuli[3]. Ex vivo proliferative responses of PKCβ − / − splenic B-cells following BCR engagement are significantly reduced incomparison with PKCβ + / + B-cells [3,4]. IL-4 (interleukin 4)promotes PKCβ − / − B-cell survival and increases the proportionof dividing cells, suggesting that accelerated death contributes toimpaired proliferative responses to BCR (B-cell antigen receptor)ligation [3].

An emerging body of evidence suggests that extrinsic signalssupport lymphocyte survival, in part, by maintaining the cellularmetabolism associated with general housekeeping functions [5–10]. Notably, naıve lymphocytes require glucose uptake andmetabolism to maintain survival and function in both ex vivo

primary cultures and in vivo [7–11]. Antigen receptor cross-linking is accompanied by increased bioenergetic and biosyntheticdemands and is characterized by a metabolic switch to aerobicglycolysis in T- and B-cells; however, the nature of this regulationand the signalling pathways involved are poorly defined inlymphocytes [12–15]. Interestingly, B-cell lymphomas exhibita high rate of aerobic glycolysis similar to that of activatedprimary B-cells [11]. In the present study, we used combinedpharmacological and genetic approaches to test directly therequirement for PKCβ in the BCR-mediated switch to glycolysis.The results described in the present paper establish a definitiverole for PKCβ in the metabolic switch to glycolysis followingBCR engagement of naıve B-cells.

MATERIALS AND METHODS

Reagents

Anti-GLUT1 (glucose transporter 1) Ab (antibody) wasfrom Research Diagnostics. Anti-PKCβ Ab was from SantaCruz Biotechnology. Anti-MEK1/2 (mitogen-activated proteinkinase/extracellular-signal-related kinase kinase 1/2) Ab wasfrom Cell Signaling Technologies. F(ab′)2 fragments of anti-Ig[goat anti-(mouse IgM)] and FITC-labelled goat anti-rabbitsecondary Ab were from Jackson ImmunoResearch Labor-atories. 2DG (2-deoxyglucose) and Go6976 were fromCalbiochem/Novabiochem. Alexa Fluor® 546-conjugated CTB(cholera toxin subunit B) was from Molecular Probes/Invitrogen.CD40L (CD40 ligand) was prepared and used as described in[16].

Abbreviations used: Ab, antibody; anti-Ig, goat anti-(mouse IgM); BCR, B-cell antigen receptor; Btk, Bruton’s tyrosine kinase; CD40L, CD40 ligand;CTB, cholera toxin subunit B; 2DG, 2-deoxyglucose; GLUT1, glucose transporter 1; HK2, hexokinase 2; PFK1, phosphofructokinase 1; PGAM1,phosphoglycerate mutase 1; PKC, protein kinase C; PKM2, pyruvate kinase M2; PPP, pentose phosphate pathway; RT, reverse transcription; xid, X-linked immunodeficiency.

1 Present address: Millennium: The Takeda Oncology Company, 40 Lansdowne Street, Cambridge, MA 02139, U.S.A.2 To whom correspondence should be addressed (email chilest@bc.edu).

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166 D. Blair, F. J. Dufort and T. C. Chiles

Mice and B-cell isolation

BALB/cByJ and xid (X-linked immunodeficiency) mice werefrom Taconic Farms. C57BL/6×129×1/SvJ and mice deficientin PKCβI/II were obtained from Dr George King (Dana FaberCancer Institute, Boston, MA, U.S.A.); PKCδ − / − mice wereobtained from Dr Thomas Rothstein (The Feinstein Institutefor Medical Research, Manhasset, NY, U.S.A.). Mice werecared for and handled at all times in accordance with NationalInstitutes of Health guidelines; vertebrate animal studies havebeen reviewed and approved by IACUC (Institutional AnimalCare and Use Committee) at Boston College. Splenic B-cellsfrom mice at 8–12 weeks of age were purified via negativeselection (Miltenyi Biotec) [8]; small dense B-cells were isolatedfollowing centrifugation through a discontinuous Percoll gradientand cultured RPMI 1640 medium plus 10% (v/v) fetal bovineserum [14]. Western blot analysis confirmed depletion of PKCβin splenic B-cells from PKCβI/II− / − mice (Supplementary FigureS1 at http://www.biochemj.org/bj/448/bj4480165add.htm).

Glucose utilization assay

B-cell cultures (106 cells/0.5 ml) were incubated with [5-3H]glucose (GE Healthcare) for 90 min. Cells (100 μl) were thenremoved and placed in 1.5 ml microcentrifuge tubes containing50 μl of 0.2 M HCl. [3H]Water was separated from unmetabolized[3H]glucose by evaporation diffusion (25 ◦C) for 48 h as describedin [14,17]. The amount of diffused and non-diffused tritium wasquantified by liquid-scintillation spectrometry and compared withvials containing [5-3H]glucose only and [3H]water only.

Indirect immunofluorescence

B-cells were centrifuged on to glass slides using a Cytospin(Thermo Electron) and incubated with 5 μg/ml Alexa Fluor®

546–CTB for 30 min at 22 ◦C. Slides were washed once withPBS and incubated in 3.7% (v/v) formaldehyde for 20 min(22 ◦C). Cells were permeabilized in 0.5 % (v/v) Triton X-100for 5 min and then blocked with 2% (w/v) BSA at 22 ◦C for30 min. Slides were incubated at 22 ◦C for 1 h with anti-GLUT1rabbit polyclonal Ab, followed by an FITC-conjugated goat anti-(rabbit IgG) as described in [14]. Following several washes withPBS, the slides were mounted with Aqua Polymount and analysedby confocal microscopy.

RT (reverse transcription)–PCR

Glycolytic gene transcripts were quantified as described by Faberet al. [18]. Total RNA was isolated using the RNeasy mini RNAisolation kit (Qiagen), following the manufacturer’s protocol.Following DNase I treatment, 2 μg of RNA was reverse-transcribed to cDNA using MMLV (Moloney murine leukaemiavirus) reverse transcriptase (Ambion). Real-time PCR wasperformed with the SYBR Green Supermix on an iCycler withiQ5 Real-time PCR detection system (Bio-Rad Laboratories).Amplification conditions were as follows: 50 ◦C for 2 min and95 ◦C for 10 min, followed by 45 cycles of 95 ◦C for 15 s and58 ◦C for 1 min. The following primers were used: PFK1_fwd,5′-TCCGAGGAAGGCGTTTTGA-3′, and PFK1_rev, 5′-TGAC-GGCTACATTGCAGTTGC-3′, for phosphofructokinase 1;PKM2_fwd, 5′-GCCGCCTGGACATTGACTC-3′, andPKM2_rev, 5′-AGCCGAGCCACATTCATTCC-3′, for pyruvatekinase M2; HK2_fwd, 5′-TGGAGATTTCTAGGCGGTTCC-3′,and HK2_rev, 5′-CATCCGGAGTTGACCTCACAA-3′, for

hexokinase 2; PGAM1_fwd, 5′-AGAGCACTGCCCTTCTGGA-AT-3′, and PGAM1_rev, 5′-TCCATGATGGCCTCTTCTGAG-3′, for phosphoglycerate mutase 1; and β2MG_fwd, 5′-CACCCGCCTCACATTGAAATA-3′, and β2MG_rev, 5′-CATGCTTAACTCTGCAGGCGT-3′, for β2-microglobulin.

RESULTS AND DISCUSSION

To test the hypothesis that PKCβ is essential in BCR-inducedglycolytic metabolism, we first evaluated the expression ofGLUT1, which is a ubiquitously expressed facilitated glucosetransporter induced on the cell surface upon B- and T-cellactivation and is necessary to promote glycolytic flux [12,14,19].Both wild-type and PKCβ − / − quiescent B-cells exhibit GLUT1expression as visualized by immunofluorescence (Figure 1A).Incubation of wild-type and PKCβ − / − splenic B-cells with anti-Ig results in increased GLUT1 expression (Figure 1A). We alsoexamined GLUT1 expression by Western blot analysis in isolatedplasma membrane fractions. GLUT1 expression increases inwild-type and PKCβ − / − B-cells following BCR engagement(Figure 1B). As a positive control for these studies, CD40Ltreatment also increases GLUT1 expression in both wild-typeand PKCβ − / − B-cells (Figure 1B).

We next examined directly whether the metabolic switch toglycolysis in response to BCR engagement on naıve B-cells isdependent on PKCβ. Anti-Ig stimulation of PKCβ − / − B-cells failto increase glycolysis above that of B-cells cultured in mediumalone, whereas wild-type B-cells exhibit increased glycolysis(Figure 2A). As a positive control, incubation with CD40L resultsin increase glycolysis in both wild-type and PKCβ − / − B-cells(Figure 2A). Consistent with these findings, wild-type splenic B-cells from BALB/cByJ mice incubated with the PKCβ inhibitorGo6976 exhibit impaired glycolytic induction following BCRengagement (Figure 2B). These results suggest that PKCβ isessential in coupling the BCR to increase glycolytic metabolism.

The immune phenotype of PKCβ − / − mice is similar to thatobserved in xid mice, which have a point mutation in thepleckstrin homology domain of Btk (Bruton’s tyrosine kinase),supporting the well-established functional co-operation within anintegrated signalling pathway between Btk and PKCβ [20,21].With this in mind, we evaluated whether a similar defect in BCR-induced glycolysis is present in B-cells from xid mice. Anti-Igstimulation of wild-type C57BL/6 B-cells increases glycolysis,whereas B-cells from xid mice exhibit a significantly lower levelof glycolytic induction (Figure 2C). Of note, we also observedimpaired BCR-mediated glycolytic flux in Btk− / − splenic B-cells (results not shown). In contrast with the immunodeficiencyobserved in PKCβ − / − mice, the novel PKC isoform PKCδ plays anon-redundant role in the regulation of B-cell tolerance [2,22]. Toevaluate whether PKCδ − / − B-cells exhibit altered glycolytic fluxin response to BCR engagement, B-cells were cultured in mediumalone or with anti-Ig (Figure 2D). In contrast with PKCβ − / −

B-cells, cross-linking the BCR on PKCδ − / − B-cells increasesglycolysis (6-fold above unstimulated B-cells), whereas anti-Igstimulated wild-type B-cells exhibit a 9-fold increase in glycolysis(Figure 2D).

To understand further how PKCβ regulates BCR-inducedglycolysis, we compared the expression of several glycolyticgenes. The results reveal a diverse impact of PKCβ-deficiencyon glycolytic gene expression. The induction of HK2 mRNAis impaired following BCR engagement in PKCβ − / − B-cells(Figure 3, panel HK2). Moreover, the increase expression of HKIIprotein in response to BCR engagement in PKCβ − / − B-cellsis impaired following BCR cross-linking (Supplementary Figure

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A role for PKCβ in BCR-induced glycolysis 167

Figure 1 Expression of GLUT1 in splenic B-cells of PKCβ − / − and PKCβ + / + mice

(A) B-cells from PKCβ + / + (wild-type) and PKCβ − / − mice were cultured in medium alone or stimulated with 10 μg/ml anti-Ig (αIg) for 12 h. B-cells were incubated with anti-GLUT1 Ab, followedby FITC-labelled goat anti-rabbit secondary Ab (upper panels); B-cells were also stained with Alexa Fluor® 546-conjugated CTB (lower panels) in order to detect plasma membrane GM1 gangliosideas described in [14]. Analysis was carried out with a Leica TCS SP2 confocal laser-scanning microscope with a ×100/0.53 numerical aperture objective. Leica confocal software V2.61 was usedto acquire the images. Images are representative of three independent experiments. (B) Parallel B-cells were cultured in the absence (M) or presence of 10 μg/ml anti-Ig (αIg) or CD40L; plasmamembranes were then isolated, detergent lysates were prepared, and equivalent amounts of protein (30 μg) were examined by Western blotting for GLUT1 expression [14,25].

S2 at http://www.biochemj.org/bj/448/bj4480165add.htm). Theinduction of PKM2 and PGAM1 mRNAs are also impairedfollowing BCR engagement in PKCβ − / − B-cells (Figure 3, panelsPKM2 and PGAM1), whereas the induction of PFK1 in PKCβ − / −

B-cells is not significantly altered compared with wild-type B-cells (Figure 3, panel PFK1).

To test the importance of glycolysis induced via cross-linkingof the BCR, we cultured B-cells in the presence and absenceof 2DG, an inhibitor of the glycolytic pathway. 2DG inhibitsHK2, the first rate-limiting enzyme of glycolysis. In controlstudies, treatment of anti-Ig-stimulated B-cells from BALB/cByJmice with 2DG reduces glycolytic flux, as expected (Table 1).Moreover, 2DG significantly reduces B-cell viability despite thepresence of anti-Ig in the culture medium (Table 1). Consistentwith these findings, B-cells treated with iodoacetamide,which inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, also exhibit decreased cell viabilitydespite signalling from the BCR (Supplementary Table S1 athttp://www.biochemj.org/bj/448/bj4480165add.htm) [23].

In the present study, we found evidence that PKCβ expressionis necessary for the metabolic switch to glycolysis followingBCR engagement of naıve B-cells. The results also suggest thatBtk is required for glycolytic induction following BCR cross-linking. The findings suggest that the BCR-mediated increasein glycolysis results, at least in part, from PKCβ-dependent up-regulation of several key glycolytic genes, including HK2 andPKM2; HK2 is the first rate-limiting enzyme of the glycolytic

Table 1 Inhibition of glycolysis reduces ex vivo splenic B-cell viability inthe presence of BCR cross-linking

Glycolysis: B-cells from control BALB/cByJ mice were cultured in medium alone for 2 h orstimulated with 10 μg/ml anti-Ig for 24 h. Glycolysis was then measured for 90 min in theabsence or presence of 1 mM 2DG as described in the Materials and methods section. Resultsare means+−S.D. of triplicate measurements. Viability: B-cells from control BALB/cByJ micewere cultured in medium alone for 2 h or stimulated with 10 μg/ml anti-Ig for 24 h in theabsence and presence of 1 mM 2DG. Viability was determined by propidium iodide staining byflow cytometry [10]. Results are representative of three independent experiments.

Glycolysis (nmol/106 cells per h) B-cell viability (%)

Treatment Without 2DG With 2DG (1 mM) Without 2DG With 2DG (1 mM)

B-cells, medium 12.9 +− 0.6 11.1 +− 0.3 91.2 89.1B-cells + anti-Ig 65.5 +− 0.3 18.5 +− 0.7 43.3 6.0

pathway. Interestingly, the induction of PFK1 gene expressionwas not significantly impaired in PKCβ − / − splenic B-cells; PFK1catalyses the committed step of glycolysis with conversion offructose 6-phosphate and ATP into fructose 1,6-bisphosphateand ADP. In contrast with the findings with PKCβ, which isnecessary for survival and growth stimulation, our results fail toimplicate PKCδ in BCR-induced glycolytic flux. PKCδ functionsas a critical negative regulator in activated B-cells inasmuch asablation of PKCδ − / − in mice is associated with a specific lossof peripheral B-cell tolerance [2,22]. Taken together, the findings

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168 D. Blair, F. J. Dufort and T. C. Chiles

Figure 2 Impaired up-regulation of glycolysis in PKCβ − / − B-cells but not PKCδ − / − B-cells after BCR cross-linking

(A) B-cells from wild-type (open bars) or PKCβ − / − (closed bars) mice were cultured in medium alone or stimulated with 10 μg/ml anti-Ig or CD40L. (B) B-cells obtained from control BALB/cByJmice were cultured in medium alone or stimulated with 10 μg/ml anti-Ig. The B-cells were also cultured in the absence (closed bars) or presence of an equal volume of DMSO (open bars) or 100 nMGo6976 (hatched bar). Note that Go6976 was dissolved in DMSO. (C) B-cells from wild-type (closed bars) or xid (open bars) mice were cultured in medium alone or stimulated with 10 μg/ml anti-Ig.(D) B-cells from wild-type (open bars) or PKCδ − / − (closed bars) mice were cultured in medium alone or stimulated with 10 μg/ml anti-Ig. In all panels, B-cells were cultured for 8.5 h and then themedium was supplemented for 90 min with [5-3H]glucose and glycolysis was measured as described in the Materials and methods section. Results are means +− S.D. of triplicate measurements,representative of three independent experiments. Cell viability was greater than 85 % for all experimental conditions.

Figure 3 Reduced expression of several glycolytic genes after BCR cross-linking on PKCβ − / − splenic B-cells

Glycolytic gene transcripts (HK2, PKM2, PFK1 and PGAM1) were measured by RT–PCR and the amounts normalized to β2-microglobulin as described in the Materials and methods section. B-cellsfrom wild-type (open bars) or PKCβ − / − (closed bars) mice were evaluated. Results are means +− S.D. of triplicate measurements, representative of three independent experiments.

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A role for PKCβ in BCR-induced glycolysis 169

suggest distinct biological roles for specific PKC isoforms (i.e.PKCβ) in regulating glucose energy metabolism.

Our findings also indicate that glycolysis is required to maintainsurvival of ex vivo B-cells stimulated via the BCR. We basethis conclusion on the use of 2DG, which inhibits HK2 andis thus widely used to evaluate the biological significance ofglycolysis in mammalian cells and recently in the differentiationof TH17 (T-helper 17) and Treg (regulatory T-) cells [24]. We areaware that inhibition of HK2 with 2DG may prevent glucose 6-phosphate metabolism by the PPP (pentose phosphate pathway).However, we have reported previously that glucose 6-phosphateflux through the PPP is minimal during the first 24 h followingBCR engagement [14]. Moreover, pre-treatment of naıve B-cells with inhibitors of the PPP do not measurably decreaseB-cell viability in response to BCR engagement (F.A. Dufort,unpublished work). Along these lines, we cannot rule out apotential secondary effect of 2DG on mitochondrial oxidativemetabolism.

It is noteworthy that cross-linking the BCR also promotesB-cell growth. At present, we cannot rule out an additionalrole for PKCβ-dependent glycolysis in the growth of B-cells,which occurs in response to BCR ligation. A major functionof glycolysis in activated lymphocytes is to support de novomacromolecular synthesis necessary for growth and proliferation[11]. Nevertheless, these results provide the first evidence of anessential role for PKCβ in the metabolic switch to glycolysisfollowing BCR engagement of naıve B-cells. Moreover, ourfindings suggest that the regulation of glycolysis by PKCβcontributes to the well-documented role for PKCβ in BCR-mediated B-cell survival. Finally, the results may providenew therapies in the form of targeting glycolytic enzymes incombination with PKCβ for immune disorders characterizedby dysregulated glycolysis and elevated PKCβ activity, such asobserved in a subset of diffuse large B-cell lymphomas [1].

AUTHOR CONTRIBUTION

Derek Blair and Fay Dufort assisted in the design of experiments,performed all of the experiments and interpreted the data.Thomas Chiles conceived of the study, assisted in the designof experiments and wrote the paper.

ACKNOWLEDGEMENTS

We thank Dr Robert Woodland for helpful discussions and Blair Bleiman for technicalassistance.

FUNDING

This work was supported by the National Institutes of Health [grant number 5R01AI074687(to T.C.C.)] and the Margaret Walsh Faculty Research Fund (to T.C.C.).

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14 Doughty, C. A., Bleiman, B. F., Wagner, D. J., Mataraza, J. M., Roberts, M. F. and Chiles,T. C. (2006) Antigen receptor-mediated changes in glucose metabolism in B lymphocytes:role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood107, 4458–4465

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25 Boyd, R. S., Adam, P. J., Patel, S., Loader, J. A., Berry, J., Redpath, N. T., Poyser, H. R.,Fletcher, G. C., Burgess, N. A., Stamps, A. C. et al. (2003) Proteomic analysis of thecell-surface membrane in chronic lymphocytic leukemia: identification of two novelproteins, BCNP1 and MIG2B. Leukemia 17, 1605–1612

Received 1 August 2012/4 September 2012; accepted 21 September 2012Published as BJ Immediate Publication 21 September 2012, doi:10.1042/BJ20121225

c© The Authors Journal compilation c© 2012 Biochemical Society

Biochem. J. (2012) 448, 165–169 (Printed in Great Britain) doi:10.1042/BJ20121225

SUPPLEMENTARY ONLINE DATAProtein kinase Cβ is critical for the metabolic switch to glycolysisfollowing B-cell antigen receptor engagementDerek BLAIR1, Fay J. DUFORT and Thomas C. CHILES2

Department of Biology, Boston College, 140 Commonwealth Avenue, 414 Higgins Hall, Chestnut Hill, MA 02467, U.S.A.

Figure S1 Expression of PKCβ in wild-type but not PKCβ − / − -deficientsplenic B-cells

Splenic B-cells from PKCβ + / + (wt) and PKCβ − / − (-/-) mice were isolated and whole-celldetergent-soluble extracts were prepared. Equivalent amounts of protein (10 μg) were thenexamined by Western blot analysis for PKCβ (upper panel) expression as described in [1]; theblot was then stripped and re-probed for expression of hsp90 (heat-shock protein 90) (lowerpanel). The molecular mass in kDa is indicated on the left-hand side.

Figure S2 Increased expression of HK2 in wild-type but not PKCβ − / −

splenic B-cells following BCR cross-linking

B-cells from PKCβ + / + (wt) and PKCβ − / − (-/-) mice were isolated and cultured in the absence(M) or presence of 10 μg/ml anti-Ig (αIg). Whole-cell detergent-soluble extracts were prepared,and equivalent amounts of protein (30 μg) were then examined by Western blot analysis forHK2 expression (upper panels) as described in [1]; the blot was then stripped and re-probedfor expression of MEK1/2 (mitogen-activated protein kinase/extracellular-signal-related kinasekinase 1/2) (lower panels). The molecular mass in kDa is indicated on the left-hand side.

REFERENCE

1 Doughty, C. A., Bleiman, B. F., Wagner, D. J., Mataraza, J. M., Roberts, M. F. and Chiles,T. C. (2006) Antigen receptor-mediated changes in glucose metabolism in B lymphocytes:role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood107, 4458–4465

Received 1 August 2012/4 September 2012; accepted 21 September 2012Published as BJ Immediate Publication 21 September 2012, doi:10.1042/BJ20121225

Table S1 Iodoacetamide (IAA)-mediated inhibition of glycolysis reducesex vivo splenic B cell viability

Glycolysis: B-cells from control BALB/cByJ mice were stimulated with 10 μg/ml anti-Ig for24 h. Glycolysis was then measured for 90 min in the absence or presence of 10 μM IAA asdescribed in the Materials and methods section of the main text. Results are means +− S.D. oftriplicate measurements. Viability: B-cells from control BALB/cByJ mice were stimulated with10 μg/ml anti-Ig for 24 h in the absence and presence of 10 μM IAA. Viability was determinedby propidium iodide staining by flow cytometry. Results are representative of three independentexperiments.

Glycolysis (nmol/106 per h) B-cell viability (%)

Treatment Without IAA With IAA Without IAA With IAA

B cells + anti-Ig 65.6 +− 0.2 25.8 +− 0.4 43.3 2.0

1 Present address: Millennium: The Takeda Oncology Company, 40 Lansdowne Street, Cambridge, MA 02139, U.S.A.2 To whom correspondence should be addressed (email chilest@bc.edu).

c© The Authors Journal compilation c© 2012 Biochemical Society