1. Supplementary Figure 1
a) Human miRNA-34a - Axl
b) Human miRNA-199a - Axl
c) Human miRNA-199b - 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

2. Supplementary Figure 2 H1299 Rko 1 2 3 1 2 3 c-Met Notch 1 HIF1 a
c-Met
Notch 1
HIF1 a
β-Actin
1: Control-miR
2: PM-miR-34a
3: PM-miR-199a
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.

3. Relative miR expression (Log Scale)
Supplementary Figure 3
a) PM-miRNA expression
H1299
0.1 10
1000
100000
Con-miR
miR-34a
miR-199a
MDA-MB-231
0.1 1 10
100
1000
Con-miR
miR-34a
miR-199a
Rko
0.1 1 10
100
1000
10000
Con-miR
miR-34a
miR-199a
HCT116
Relative miR expression (Log Scale)
10000
100
0.1
Con-miR
miR-34a
miR-199a
b) AM-miRNA expression
A549
MCF7
Colo 320
1.2
1.2
1.2
1.0
1.0
1.0
0.8
0.8
0.8
0.6
0.6
0.6
Relative miR expression
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
0.0
Con-miR
miR-34a
Con-miR
miR-34a
Con-miR
miR-34a
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.

4. Relative miR-34a expression Relative miR-34a expression (Log scale)
Supplementary Figure 4 a)
NSCLC
BRC
CRC
2000
4000
6000
8000
10000
H1299
Calu-1
H522
LXF289
H596
Calu-3
H520
SK-MES 1
A549
H1395
H460
0.1 1 10
100
1000
2000
4000
6000
Rko
HCT116
Colo 320
HT-29
WiDr
Colo206f
SW480
HCT15
Geo
Relative miR-34a expression
Relative miR-34a expression (Log scale)
Relative miR-34a expression
MDA- MB-231
MCF-7 b)
NSCLC
BRC
CRC
250 6
0.1 1 10
100
1000
Rko
HCT116
SW480
Colo 320
HT-29
HCT15
WiDr
Geo
Colo 206f
200
Relative miR-199a expression
150
Relative miR-199a expression 4
Relative miR-199a expression(Log scale)
100 2 50
H1299
Calu-1
H522
H596
Calu-3
H520
A549
H460
MDA- MB-231
LXF289
H1395
MCF-7
SK-MES-1
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

5. Relative Axl mRNA expression Relative Axl mRNA expression(Log scale)
Supplementary Figure 5
NSCLC
BRC
CRC a) 50
100
150
200
250
300
0.1 1 10
100
1000
MCF-7
MDA- MB-231
10000
400
800
1200
1600
Rko
HCT116
Colo 320
HT-29
WiDr
Colo206f
SW480
HCT15
Geo
Relative Axl mRNA expression
Relative Axl mRNA expression(Log scale)
Relative Axl mRNA expression
H1299
Calu-1
H522
H596
Calu-3
H520
A549
H1395
H460
LXF289
SK-MES 1 b)
NSCLC
BRC
CRC
0.0
0.4
0.8
1.2
H1299
Calu-1
H522
LXF289
H596
Calu-3
H520
SK-MES 1
A549
H1395
H460 1 2 3
MCF-7
MDA- MB-231
1.6
1.2
0.8
Axl Protein ratio: (Axl/Actin)
Axl Protein ratio: (Axl/Actin)
Axl Protein ratio: (Axl/Actin)
0.4
0.0
Rko
SW480
HT-29
WiDr
Geo
HCT15
Colo 320
HCT116
Colo206f c)
Axl
β-Actin
130 kDa
43 kDa
1)Calu 3; 2)A549; 3)H460; 4)H520; 5)LXF-289; 6) H1395; 7)Calu1; 8)H596; 9)H522; 10)SK-MES-1 11)H1299; 12)MDA-MB-231; 13)MCF-7; 14)Rko; 15)HCT116; 16)SW480; 17)Colo 320; 18) HT-29; 19)HCT15 20)WiDr; 21)Geo; 22) Colo 206f
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.

6. a) Axl-protein vs invasion b) miR-34a-expression vs invasion
Supplementary Figure 6
a) Axl-protein vs invasion
b) miR-34a-expression vs invasion
c) miR-34a-expression vs Axl-protein
Axl protein expression
Invasive ability
R= 0.41; p=0.03
Log[miR-34a expression]
Axl protein expression
R= -0.81; p<0.01
Log[miR-34a expression]
Invasive ability
R= -0.75; p<0.01
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).

7. Supplementary Figure 7 a) b)
H1299
Rko 14 14 12 12 10 10 * * 8 * 8 6
Percent of cells migrated 6
Percent of cells migrated 4 4 * 2 2
Control-miR
miR-34a
miR-199a
Control-miR
miR-34a
miR-199a
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 (*p0.05)

8. Supplementary Figure 8
Axl (NM_ ): Chromosome 19
miR-34a (MI000268) : Chromosome 1
miR-199a1-5p (MI ): Chromosome 19
miR-199a2-5p (MI ): Chromosome 1
miR-199b-5p (MI ) : 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