Supplementary Figure 1

b) Human miRNA-199a - Axl c) Human miRNA-199b - Axl

a) Human miRNA-34a - Axl

Predicted secondary structures (Pic Tar predicitions) of Axl and miR-34a and –199a/b:a,b,c) Secondary structures between Axl ( 3’UTR 24 to 50 nt) and miR-34a, Axl ( 3’UTR 25 to 56 nt) and miR-199a/b. Green-sequences represent the miR; a Red-sequence represents the Axl-3’UTR

Supplementary Figure 2

H1299 and Rko cells were transfected either with miR-34a or -199a. After 48h, protein was isolated and Western blot analysis was performed for c-Met, Notch 1 and HIF1a. β-Actin served as internal control.

c-Met

HIF1 a

β-Actin

Notch 1

1: Control-miR 2: PM-miR-34a3: PM-miR-199a

1 2 3

H1299

1 2 3

Rko

Supplementary Figure 3

0.00.20.40.60.81.01.2

0.00.20.4

0.60.81.01.2

0.00.20.40.60.81.01.2

a) PM-miRNA expression

Rel

ativ

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iR e

xpre

ssio

n

(Log

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-miR

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-34a

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Rko

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H1299

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MDA-MB-231

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HCT116

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b) AM-miRNA expression

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-miR

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-miR

miR

-34a

A549 MCF7 Colo 320

Expression levels of either PM-miR-34a and -199a:H1299, MDA-MB-231, Rko and HCT116 cells were transfected either with PM-miR-34a or PM-miR-199°, and A549, MCF-7, Colo 320 cells were transfected with AM-miR-34a. After 48h, miR expression levels were determined with Real-Time PCR.

Supplementary Figure 4

0.11

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NSCLC BRC CRC

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b)

NSCLC BRC CRC

miR-34a- and miR-199a-endogenous expression:Relative miR-34a and miR-199a levels in the panel of NSCLC, BRC and CRC cell lines evaluated by Real-Time PCR

Rel

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NSCLC BRC CRCa)

b) NSCLC BRC CRC

Supplementary Figure 5

Axl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

β-Actin

1)Calu 3; 2)A549; 3)H460; 4)H520; 5)LXF-289; 6) H1395; 7)Calu1; 8)H596; 9)H522; 10)SK-MES-111)H1299; 12)MDA-MB-231; 13)MCF-7; 14)Rko; 15)HCT116; 16)SW480; 17)Colo 320; 18) HT-29; 19)HCT1520)WiDr; 21)Geo; 22) Colo 206f

c)

Axl endogenous expression in NSCLC, BRC and CRC cell lines:a) Relative Axl expression in the panel of NSCLC, BRC and CRC cell lines evaluated by Real-Time PCR, β-Actin served as internal control. b) Axl densitometric Western blot analysis. The density ratio of Axl/-Actin is represented as a bar graph. c) Axl protein amounts (Western blot analysis, β-Actin served as internal control) in NSCLC, BRC and CRC cell lines.

130 kDa

43 kDa

Rel

ativ

e A

xl m

RN

A e

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Axl

pro

tein

exp

ress

ion

R= -0.81; p<0.01

Log[miR-34a expression]

Inva

sive

ab

ilit

y

R= -0.75; p<0.01

Axl protein expression

Inva

sive

ab

ilit

y

R= 0.41; p=0.03

c) miR-34a-expression vs Axl-proteina) Axl-protein vs invasion b) miR-34a-expression vs invasion

Supplementary Figure 6

Correlations between invasion, miR34a- and Axl-endogenous expression in NSCLC cells:a) Axl protein vs invasion, b) miR34a-expression vs invasion, c) miR-34a expression vs Axl protein in a panel of NSCLC cell lines (p-value is from Spearman’s correlation test).

Supplementary Figure 7

H1299

02468

101214

Control-miR miR-34a miR-199aPer

cen

t of

cel

ls m

igra

ted

Rko

02468

101214

Per

cen

t of

cel

ls m

igra

ted

Control-miR miR-34a miR-199a

*

*

* *

a) b)

miR-34a and -199a inhibits migration in Boyden chambers:H1299 and Rko cells were transfected and plated in the upper chamber, and after 12h top and invaded cells were counted as described for matrigel assays in materials and methods. a,b) both the miRs (miR-34a and 199a) significantly reduced migration of H1299 and Rko cells. Data represented as the percentage of invading cells, as mean ± SD of four replicates (*p0.05)

miR-34a (MI000268) : Chromosome 1

miR-199a1-5p (MI0000242): Chromosome 19

Supplementary Figure 8

Axl (NM_021913 ): Chromosome 19

miR-199b-5p (MI0000282) : Chromosome 9

Methylation status of Axl, miR-34a and miR-199a/b promoters:2000 bp or more bases were taken from the TSS or upstream sequence from the pre-miR chromosomal location for CpG islands. Horizontal small red lines represent the CpG island, and a thick black underline represents the region taken for methylation analysis

miR-199a2-5p (MI0000281): Chromosome 1