Fig. S1 1 Oxygen consumption rate (pmol/sec/10⁶cells) shLKB1#1 control * untreated 25 μM...

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Fig. S1 1 Oxygen consumption rate (pmol/sec/10⁶cells) shLKB1#1 control * untreated 25 μM erlotinib H358 Vector LKB1 Oxygen consumption rate (pmol/sec/10⁶cells) untreated 25 μM erlotinib H460 * Figure S1. Erlotinib inhibits oxygen consumption rate (OCR) in LKB1- deficient cells. A, OCR of LKB1 stable knockdown and non-targeted shRNA control H358 cells. B, OCR of H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were treated with erlotinib for 24 h. Data are mean ± SEM (n=3). *; p<0.05, untreated/erlotinib. A B

Transcript of Fig. S1 1 Oxygen consumption rate (pmol/sec/10⁶cells) shLKB1#1 control * untreated 25 μM...

Fig. S1

1

Oxy

gen

cons

umpti

on ra

te

(pm

ol/s

ec/1

0⁶ce

lls)

shLKB1#1control

*

untreated25 μM erlotinib

H358

Vector LKB1

Oxy

gen

cons

umpti

on ra

te

(pm

ol/s

ec/1

0⁶ce

lls)

untreated25 μM erlotinib

H460

*

Figure S1. Erlotinib inhibits oxygen consumption rate (OCR) in LKB1-deficient cells. A , OCR of LKB1 stable knockdown and non-targeted shRNA control H358 cells. B, OCR of H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were treated with erlotinib for 24 h. Data are mean ± SEM (n=3). *; p<0.05, untreated/erlotinib.

A B

A

Spec

ific

gro

wth

rate

(h⁻¹)

H460

Vector LKB1

B

Fig. S2

Figure S2. Effect of erlotinib on growth and glycolytic metabolism of H460 cells. A , Effect of treatment with erlotinib on cell growth rate in H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were seeded in 96-well plates. After 24 h, cells were treated with the indicated concentrations of erlotinib and growth was monitored for 72 h. B, Glucose uptake rate and lactate production rate in H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were grown for 72 h in the presence of treatments.

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Vector LKB1

Glu

cose

upt

ake

rate

mol

/10⁶

cel

ls/h

)La

ctat

e pr

oduc

tion

rate

mol

/10⁶

cel

ls/h

)

Vector LKB1

untreated15 μM erlotinib

untreated15 μM erlotinib

H460

Fig. S3

p-AMPKα

AMPKα

p-ACC

ACC

control

erlotinib

0 3 6 24 hours- + + +

shLKB1 #1

0 3 6 24- + + +

1 0.79 1.22 2.32 1 1.1 0.83 2.06

1 0.91 0.6 0.8 1 0.99 1 1.63

H358

actin

Figure S3. Erlotinib-induced activation of AMPKα in LKB1-deficient H358 cells. Erlotinib activated AMPKα and induced phosphorylation of ACC in shLKB1 H358 cells. Following treatment with 0.5 μM erlotinib for the indicated time, cell lysates were analyzed by Western blotting. The expression of phosphorylated AMPKα was calculated as the ratio of phosphorylated AMPKα to total AMPKα protein expression using densitometric analysis. The ratio of phosphorylated to total ACC was calculated similarly. All expression ratios were normalized to the untreated group.

Fig. S4

control shLKB1#1

0.5 µM erlotinib- +

A

- +

p-mTOR (Ser2448)

p-AMPKα

mTOR

AMPKα

p-p70S6K (Thr389)

p-Akt (Ser473)

Akt

p-Erk

B

H358 A549

- -

vector LKB1

p70S6K (Thr389)

P-4EBP1 (Thr37/46)

P-4EBP1

actin

Erk

+ + - -

vector LKB1

+ +

H460

0.5 µM erlotinib

p-mTOR (Ser2448)

p-AMPKα

mTOR

AMPKα

p-p70S6K (Thr389)

p-Akt (Ser473)

Akt

p-Erk

p70S6K (Thr389)

P-4EBP1 (Thr37/46)

P-4EBP1

actin

Erk

Figure S4. Erlotinib-induced activation of AMPKα selectively inhibits mTOR signaling pathway in LKB1-deficient cells. Erlotinib activated AMPKα and blocked mTOR pathway signaling in A, shLKB1 H358 cells and B, LKB1-non expressing A549 and H460 cells. After 24 h of 0.5 μM erlotinib treatment, cell lysates were analyzed by Western blotting using specific antibodies for the indicated proteins. Blockade of mTOR activity following erlotinib treatment resulted in decreased phosphorylation of p70S6K and p-4EBP1 in LKB1 deficient cells.

Fig. S5

control shLKB1#1

0.5 µM erlotinib- +

shLKB1#2

A

- + - +

PDK4

Actin

Calu-6 A549

0.5 µM erlotinib - -

vector LKB1

PDK4

Actin

+ +

control shLKB1#1

0.5 µM erlotinib- + - +

PDK4

Actin

H358 H460

0.5 µM erlotinib - -

vector LKB1

PDK4

Actin

+ +

Figure S5. Levels of PDK4 expression is not changed by erlotinib treatment in both LKB1-deficient and overexpressing NSCLC cells. A, Levels of PDK4 expression in LKB1 stable knockdown and non-targeted shRNA control Calu-6 and H358 cells. B, Levels of PDK4 expression in A549 and H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were treated with 0.5 μM erlotinib for 24 h and cell lysates were analyzed by Western blotting.

B