0

5

10

15

20

25

30

35

40

45

50

CTR ESOM

An

nex

in-V

+ ce

lls (%

)

DMSO

DPI

A

Supplemental Figure 1. (A) Preincubation of Me30966 cells with the NADPH oxidase inhibitor DPI abrogated the apoptosis induced by esomeprazole (160 M). (B) FACS analysis of Me30966 cells stained with LysoSensor Green DND-189 shows that ESOM-treated cells (grey hystogram) carry lysosomes with a more alkaline lumen pH than untreated cells (empty hystogram). (C) Me30966 cells treated with esomeprazole (160 M) for 8 hours were analysed by LSCM for expression pattern of endogenous LC3. The figure shows an increase in the numbers of cells with punctuate LC3 fluorescence in ESOM-treated cells, quantified in the chart below. (D, E) Me30966 cells were cultured in presence of different inhibitors of the lysosomal function and treated or not with ESOM or with EBSS for 4 hours. The ratio LC3-II/actin is reported and indicates that ESOM slows down the autophagic flux.

B

DAPI LC3

CTR ESOM

Lysosensor Green DND-189

C

0

10

20

30

40

50

60

70

80

CTR ESOMC

ells

wit

h L

C3+

do

ts /

tota

l cel

ls (

%)

0.33

1.80

1.40

2.10

0.59

1.60

1.10

1.74

0.00

0.50

1.00

1.50

2.00

2.50

Medium E64d/PepA Vinblastine E64d/PepA/Vinblastine

LC3

II/A

ctin

CTR

ESOM

D E

0 2h 4h 8h 2h 4h 8h

EBSS EBSS + E64d/PepA

0.33 0.27 0.57 0.79 0.73 1.04 1.47

A

Supplemental Figure 2. A) Knock-down of ATG5 and BECN1 expression was assessed by WB analysis in WM793 cells. B) The effects of Atg5 and Beclin-1 knockdown by siRNA on cell death is shown on Me30966 cells treated with ESOM (160 M). C) Clonogenic survival assay performed on three melanoma cell lines treated with ESOM (160 M) for 24 hours. Colonies were counted 15 days after removing dead cells. D) Analysis of basal autophagy levels in colonies collected at the end of the clonogenic survival assay. Basal autophagy is expressed as the ratio of LC3-II/actin detected by WB.

B

Atg5In

terfe

rin

siSCR 2

nM

siSCR 5

nM

siATG5

2 nM

siATG5

5 nM

300 m

siCTR siBeclin-1 siAtg5

Beclin-1

-actin

Inte

rferin

siSCR 2

nM

siSCR 5

nM

siBECN-1

2 n

M

siBECN-1

5 n

M

-actin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Me30966 Me501 WM793

rati

o L

C3

-II/

ac

tin

CTR

ESOM

0

50

100

150

200

250

300

350

400

450

Me30966 Me501 WM793

nu

mb

er

of

co

lon

ies

CTR

ESOM

C D