Supplementary Figure 1
This presentation is the property of its rightful owner.
Sponsored Links
1 / 8

Supplementary Figure 1 PowerPoint PPT Presentation


  • 99 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Supplementary Figure 1

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Supplementary figure 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


Supplementary figure 1

Supplementary Figure 2

H1299

Rko

1 2 3

1 2 3

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.


Supplementary figure 1

Rko

MDA-MB-231

HCT116

10000

100000

1000

1000

100

1000

100

10

10

10

1

1

0.1

0.1

0.1

miR-34a

miR-34a

miR-34a

Con-miR

Con-miR

Con-miR

miR-199a

miR-199a

miR-199a

Supplementary Figure 3

a) PM-miRNA expression

H1299

10000000

1000000

10000

Relative miR expression (Log Scale)

100

0.1

miR-34a

Con-miR

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

miR-34a

miR-34a

miR-34a

Con-miR

Con-miR

Con-miR

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 1

1000

100

10

1

0.1

Geo

Rko

WiDr

HT-29

SW480

HCT15

HCT116

Colo 320

Colo 206f

10000

6000

1000

8000

4000

6000

100

4000

2000

10

2000

0

0

1

Rko

Geo

WiDr

HT-29

HCT15

SW480

Colo206f

A549

H460

H522

H596

H520

0.1

Colo 320

HCT116

Calu-1

H1395

H1299

Calu-3

LXF289

SK-MES 1

Supplementary Figure 4

a)

NSCLC

BRC

CRC

Relative miR-34a expression (Log scale)

Relative miR-34a expression

Relative miR-34a expression

MDA-MB-231

MCF-7

NSCLC

BRC

CRC

b)

250

6

200

4

Relative miR-199a expression(Log scale)

Relative miR-199a expression

Relative miR-199a expression

150

100

2

50

0

0

MDA-MB-231

H460

A549

H522

H596

H520

H1299

H1395

Calu-3

Calu-1

MCF-7

LXF289

SK-MES-1

miR-34a- andmiR-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


Supplementary figure 1

300

10000

250

1600

1000

200

1200

100

150

800

10

100

400

1

50

0.1

0

0

Rko

WiDr

Geo

HT-29

SW480

HCT15

Colo206f

Colo 320

MDA-MB-231

HCT116

MCF-7

1.2

3

0.8

2

0.4

1

0.0

0

A549

MDA-MB-231

H460

H522

H596

H520

H1299

Calu-1

H1395

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

Calu-3

MCF-7

LXF289

SK-MES 1

Axl

β-Actin

Supplementary Figure 5

NSCLC

BRC

CRC

a)

Relative Axl mRNA expression(Log scale)

Relative Axl mRNA expression

Relative Axl mRNA expression

A549

H460

H596

H522

H520

Calu-1

H1395

H1299

Calu-3

LXF289

SK-MES 1

NSCLC

BRC

CRC

b)

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

Geo

WiDr

HT-29

HCT15

SW480

Colo206f

HCT116

Colo 320

c)

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-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

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.


Supplementary figure 1

R= -0.75; p<0.01

R= 0.41; p=0.03

Invasive ability

Invasive ability

Log[miR-34a expression]

Axl protein expression

R= -0.81; p<0.01

Axl protein expression

Log[miR-34a expression]

Supplementary Figure 6

a) Axl-protein vs invasion

b) miR-34a-expression vs invasion

c) miR-34a-expression vs Axl-protein

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 1

Supplementary Figure 7

a)

b)

H1299

Rko

14

14

12

12

10

10

*

*

8

8

*

6

6

Percent of cells migrated

Percent of cells migrated

4

4

*

2

2

0

0

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)


Supplementary figure 1

Supplementary Figure 8

Axl (NM_021913 ): Chromosome 19

miR-34a (MI000268) : Chromosome 1

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

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

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


  • Login