GLUT1 GLUT3 HK1 HK2 HK3 PFK PK-M2 LDH PDH PDK20

1

2

3R

elat

ive

Prot

ein

Qua

ntifi

catio

n

U87-BevS

U87-BevR

**

*

* P<0.05, ** P<0.01, *** P<0.001

Supplemental Figure 1. Quantification of glycolytic enzyme protein levels in bevacizumab-resistant versus responsive xenografts. Shown are quantifications of band intensities from the western blot shown in Figure 2a (n=5/group). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value. Expression of GLUT3 and LDH increased (P=0.008) in U87-BevR, while expression of HK3 decreased (P=0.03). (Wilcoxon-Mann Whitney test).

**

Supplemental Figure 2. Transduction of U87-BevR cells with GLUT3 shRNA. Shown is western blot for GLUT3 and GAPDH for U87-BevR cells, U87-BevR cells transduced with empty vector (pLK0.1), and U87-BevR cells transduced with GLUT3-targeting shRNA sequence #15 and #16. Successful knockdown with sequence #15 serves to verify the specificity of our GLUT3 antibody. However, this knockdown proved to not be durable and only occurred with one of two shRNA sequences, suggesting a dependence of U87-BevR cells on GLUT3 for their survival.

U87

-Bev

R

U87

-Bev

R/p

LK0.

1

U87

-Bev

R/

GLU

T3 s

hRN

A #1

5

U87

-Bev

R/

GLU

T3 s

hRN

A #1

6

0

1

2

3

4

µm

ol g

luco

se/

mill

ion

cells

U87-BevS

U87-BevR

IgG

Bevacizumab Treatment:

Intracranial Xenograft:

IgG

Bevacizumab

Supplemental Figure 3. Bevacizumab treatment increases intratumoral glucose levels in bevacizumab-resistant intracranial xenografts. Shown are quantifications of intratumoral glucose in bevacizumab-resistant versus responsive intracranial xenografts (n=4/group with 3 technical replicates). Increased intratumoral glucose occurred with bevacizumab treatment of U87-BevR intracranial xenografts (P=0.03), but not with bevacizumab treatment of U87-BevS intracranial xenografts (P=0.3) (Wilcoxon-Mann Whitney test) . For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value.

*

0.0

0.1

0.2

0.3

0.0

0.2

0.4

0.6

0.8

HIF

-1α

ban

d in

tens

ity n

orm

aliz

ed to

GA

PD

HG

LUT3 band intensity norm

alized to GA

PD

H

Supplemental Figure 4. Quantification of HIF-1α and GLUT3 levels in bevacizumab-resistant versus responsive intracranial xenografts. Shown are quantifications of band intensities from western blot in Figure 2b. Decreased HIF-1α expression occurred with bevacizumab treatment of U87-BevS intracranial xenografts (P=0.03), and increased GLUT3 expression occurred with bevacizumab treatment of U87-BevR intracranial xenografts (P=0.03) (Wilcoxon-Mann Whitney test). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value.

U87-BevS

U87-BevR

IgG

Bev IgG

Bev Treatment:

Intracranial Xenograft:

*

* P<0.05, ** P<0.01, *** P<0.001

GLUT3

HIF-1α

Protein:

HIF-1α

GLUT3

HIF-1α

GLUT3

U87-BevS

U87-BevR

IgG

Bev IgG

Bev

*

Supplemental Figure 5. HIF-1α inhibitor does not alter GLUT1 expression in bevacizumab-resistant cells. Treatment of cultured U87-BevS and U87-BevR cells with 10 µM YC-1 did not alter GLUT1 protein expression in U87-BevR and U87-BevS cells regardless of oxygen levels or glucose concentration.

Time (hours)

YC-1

Glucose (g/L) 4.5 0.3 0.3 0.3 4.5 0.3 0.3 0.3 4.5 0.3 0.3 0.3 4.5 0.3 0.3 0.3

SF7300-IgG SF7300-Bev20

25

30

35

40

45

0

2

4

6

8

10

HIF

-1α

Mea

n Fl

uore

scen

ce

HIF-1αGLUT3 G

LUT3 M

ean Fluorescence

Supplemental Figure 6. Unchanged HIF-1α and GLUT3 expression in a bevacizumab-responsive patient-derived xenograft treated with bevacizumab. (a) shown are representative GLUT3 (red) and Hif-1α (green) immunostainings of intracranial bevacizumab responsive patient-derived SF7300 xenografts. (b) shown are quantifications of these immunostainings done using Image J, which revealed that neither HIF-1α (P=0.9) nor GLUT3 (P=0.5) expression increased with bevacizumab treatment of intracranial SF7300 xenografts (n=4 tumors/group) (Wilcoxon-Mann Whitney test).

b a S

F730

0-

IgG

GLUT3 HIF-1α mergedDAPI

SF7

300-

B

ev

Proton Leak ATP Production0.0

0.1

0.2

OC

R (p

mol

min

/cel

l)

U87-BevS

U87-BevR

Basal Respiration Spare Respiratory Capacity0.0

0.1

0.2

OC

R (p

mol

min

/cel

l)

U87-BevS

U87-BevR

a

c

b

d

* P<0.05, ** P<0.01, *** P<0.001

* **

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0 20 40 60 80

OC

R (p

mol

/min

/Cel

l)

Time (min)

Mitochondrial Respiration

A B C U87-BevR

U87-BevS

Supplemental Figure 7. Detailed results of oxygen consumption rate analysis of cultured U87-BevS vs. U87-BevR cells. Shown is detailed analysis of results presented in Figure 6c. As with Figure 6c, n=46 samples/cell type/time point. (a) Figure 6c shown again - oxygen consumption rates of cultured U87-BevS vs. U87-BevR cells are shown after treatment with four different mitochondrial inhibitors per Seahorse protocol: oligomycin at 18 minutes, FCCP at 36 minutes, and Antimycin A+Rotenone at 54 minutes. (b) schematic showing how to extrapolate basal respiration, ATP production, proton leak, maximal respiration, and spare capacity values from OCR curves like that shown in (a). (c) quantification of basal respiration and spare respiratory capacity derived from curves in (a) using Seahorse software revealed decreased basal respiration in U87-BevR versus U87-BevS cells (P=0.03), with no change in spare respiratory capacity (P=0.8). (d) quantification of proton leak and ATP production derived from curves in (a) using Seahorse software revealed decreased ATP production in U87-BevR versus U87-BevS cells (P=0.008) with no change in proton leak (P=0.3). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value. Analysis done using Wilcoxon-Mann Whitney test.

COXIV Cytochrome C HSP600.0

0.5

1.0

Rel

ativ

e Pr

otei

n Q

uant

ifica

tion

Mitochondrial Western Quantification

U87-BevS

U87-BevR

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e H

SP

60 g

ene

expr

essi

on

norm

aliz

ed to

U87

-Bev

S

b a

*

* P<0.05, ** P<0.01, *** P<0.001

Supplemental Figure 8. Mitochondrial gene expression in bevacizumab-resistant versus responsive xenografts. (a) shown are quantifications of band intensities from the western blot in Figure 4e, which revealed unchanged CoxIV (P=0.4) and cytochrome C (P=0.2) protein and decreased HSP60 protein (P=0.03) in subcutaneous U87-BevR relative to U87-BevS (Wilcoxon-Mann Whitney test). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value. (b) qPCR revealed decreased HSP60 transcription in subcutaneous U87-BevR vs. U87-BevS xenografts (n=4/group) (P=0.003; student’s t-test). For dot plot, the horizontal line represents the mean and vertical line represents the standard deviation.

HSP60 gene expression

U87-BevR

**

* P<0.05, ** P<0.01, *** P<0.001

Supplemental Figure 9. Effect of hypoxia on intracellular triglycerides. Shown are intracellular triglyceride accumulations in U87-BevS cells after normoxia and hypoxia treatment. There was no signfiicant difference between these values (P=0.3; Wilcoxon-Mann Whitney test). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value.

Normoxia Hypoxia32

34

36

38

40

42

U87-BevS

RFU

Intracellular Triglyceride Levels

Supplemental Figure 10. Engineering U87-BevS cells to overexpress GLUT1 or GLUT3. (a) GLUT3 expression after transducing U87-BevS cells with empty vector (EV) or a GLUT3-expressing vector. GLUT3 expression in U87-BevS/GLUT3 was so upregulated that a more prolonged exposure was needed to visualize the U87-BevS/EV GLUT3 band compared to the U87-BevS/GLUT3 GLUT3 band. (b) GLUT1 expression after transfecting U87-BevS cells with empty vector (EV) or a GLUT1-expressing plasmid at varying DNA:lipid compound ratios (ratio in parentheses). The 6:1 ratio was used for data generated in Figure 8e.

U87

-Bev

S/E

V

U87

-Bev

S/G

LUT1

(3:1

)

U87

-Bev

S/G

LUT1

(3:2

)

U87

-Bev

S/G

LUT1

(6:1

) b a

GLUT3

GAPDH

0% 25% 50% 75% 100%

G1/G0

S

G2/M

Supplemental Figure 11. Cell cycle analysis of U87-BevS/GLUT3 vs. U87-BevS/E V c e l l s i n v a r i a b l e g l u c o s e concentrations. Shown is cell cycle distribution of U87-BevS/GLUT3 vs. U87-BevS/EV cells in (a) 0.1, (b) 0.3, and (c) 4.5 g/L glucose, as assessed by propidium iodide labeling. After 48 hours, decreasing glucose caused cells to accumulate in the G1/G0 phase and out of S phase (P<0.0001) while GLUT3 overexpression allowed escape out of G1/G0 phase (P=0.0001) and into S phase (P=0.003) with no interaction between these t w o v a r i a b l e s (P = 0 . 4 ) ( A N O VA ) . Approximately 10,000 cells were flow-analyzed in four replicates with error bars representing standard deviations between replicates.

U87-BevS/EV 24 hour

U87-BevS/EV 48 hour

U87-BevS/GLUT3 48 hour

U87-BevS/GLUT3 24 hour

0.1 g/L glucose

0% 25% 50% 75% 100%

G1/G0

S

G2/M

0.3 g/L glucose

4.5 g/L glucose

0% 25% 50% 75% 100%

G1/G0

S

G2/M

U87-BevS/EV 24 hour

U87-BevS/EV 48 hour

U87-BevS/GLUT3 48 hour

U87-BevS/GLUT3 24 hour

U87-BevS/EV 24 hour

U87-BevS/EV 48 hour

U87-BevS/GLUT3 48 hour

U87-BevS/GLUT3 24 hour

b

a

c

Proton Leak ATP Production0.00

0.02

0.04

0.06

OC

R (p

mol

min

/cel

l)

U87-BevS/EV

U87-BevS/GLUT3

Basal Respiration Spare Respiratory Capacity0.00

0.02

0.04

0.06

0.08

OC

R (p

mol

min

/cel

l)

U87-BevS/EV

U87-BevS/GLUT3

d

b

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0 20 40 60 80

OC

R (p

mol

/min

/cel

l)

Time (min)

Mitochondrial Respiration

U87-Bevs/GLUT3

U87-BevS/EV A B C

* P<0.05, ** P<0.01, *** P<0.001

a

*** *** c

Supplemental Figure 12. Detailed results of oxygen consumption rate analysis of cultured U87-BevS/GLUT3 vs. U87-BevS/EV cells. Shown is detailed analysis of results presented in Figure 9b. As with Figure 9b, n=46 samples/cell type/time point. (a) Figure 9b shown again - oxygen consumption rates (OCRs) of cultured U87-BevS/GLUT3 vs. U87-BevS/EV cells are shown after treatment with four different mitochondrial inhibitors per Seahorse protocol: oligomycin at 18 minutes, FCCP at 36 minutes, and Antimycin A+Rotenone at 54 minutes. (b) schematic showing how to extrapolate basal respiration, ATP production, proton leak, maximal respiration, and spare capacity from OCR curves like that shown in (a). (c) quantification of basal respiration and spare respiratory capacity values derived from curves in (a) using Seahorse software. Compared to U87-BevS/EV cells, U87-BevS/GLUT3 cells had reduced basal respiration (P<0.0001) with unchanged spare respiratory capacity (P=0.5). (d) quantification of proton leak and ATP production derived from curves in (a) using Seahorse software. Compared to U87-BevS/EV cells, U87-BevS/GLUT3 cells had unchanged proton leak (P=0.2) and reduced ATP production (P<0.0001). Analyses were done using Wilcoxon-Mann Whitney test. For box and whiskers plots, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value.

Supplemental Figure 13. GLUT3 upregulation does not drive tumor cell migration. Shown are percent closure from scratch assays in U87-BevS/EV and U87-BevS/GLUT3 cDNA. No significant differences between the two cell types were identified in scratch closure at any of the assessed time points (P=0.6-0.8). For box and whiskers plot, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum value.

Time:

4.5 g/L 0.3 g/L 0.1 g/L 4.5 g/L 0.3 g/L 0.1 g/L 4.5 g/L 0.3 g/L 0.1 g/L0

20

40

60

80

100

Per

cent

Clo

sed

(rel

ativ

e to

0 h

ours

)

U87-BevS/EVU87-BevS/GLUT3

0 hours

6 hours

12 hours

Glucose concentration:

0 24 48 720

5

10

Time (hours)

Fold

cha

nge

GLU

T3

expr

essi

on v

s. 0

hou

rs U87-BevS - CTLU87-BevS - GSK

0 24 48 720

5

10

Time (hours)

Fold

cha

nge

GLU

T3

expr

essi

on v

s. 0

hou

rs U87-BevR - CTLU87-BevR - GSK

Supplemental Figure 14. Effect of GSK inhibitor on GLUT3 transcription. Shown are RT-PCR results measuring GLUT3 transcription from (a) U87-BevS and (b) U87-BevR cells treated, as in Figure 7c, with 10 µM glycogen synthase kinase-3β inhibitor GSK-3 IX (“GSK”) or, as a control, an equivalent dilution of the DMSO vehicle used to dilute the GSK-3 IX (“CTL”). After 72 hours, GSK-3 IX lowered GLUT3 transcription in U87-BevS (P=0.0004) and U87-BevR (P=0.02) cells (n=3/time point/cell type; student’s t-test). For dot plot, the horizontal line represents the mean and vertical line represents the standard deviation.

*** *

* P<0.05, ** P<0.01, *** P<0.001

b a

Supplemental Figure 15. Effect of GLUT3 overexpression on tumor proliferation with and without bevacizumab treatment. Shown are Ki-67 labeling indices for the tumors in Figure 9d (n=6/group). Bevacizumab treatment lowered (P=0.002) and GLUT3 overexpression raised (P=0.01) Ki-67 labeling, with no interaction between these two variables (P=0.2; ANOVA).

* P<0.05, ** P<0.01, *** P<0.001

0.0%

10.0%

20.0%

30.0%

40.0%

IgG Bev IgG Bev

EV GLUT3 U87-BevS/EV U87-BevS/GLUT3

Ki-6

7 la

belin

g (%

)