Presentation on theme: "Supplementary Figure 1 a) Human miRNA-34a - Axl"— Presentation transcript:
1 Supplementary Figure 1a) Human miRNA-34a - Axlb) Human miRNA-199a - Axlc) Human miRNA-199b - AxlPredicted 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-MetNotch 1HIF1 aβ-Actin1: Control-miR2: PM-miR-34a3: PM-miR-199aH1299 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 3a) PM-miRNA expressionH12990.1101000100000Con-miRmiR-34amiR-199aMDA-MB-2310.11101001000Con-miRmiR-34amiR-199aRko0.1110100100010000Con-miRmiR-34amiR-199aHCT116Relative miR expression (Log Scale)100001000.1Con-miRmiR-34amiR-199ab) AM-miRNA expressionA549MCF7Colo 3188.8.131.52.01.01.00.80.184.108.40.206.6Relative miR expression0.40.40.40.20.20.20.00.00.0Con-miRmiR-34aCon-miRmiR-34aCon-miRmiR-34aExpression 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 4a)NSCLCBRCCRC200040006000800010000H1299Calu-1H522LXF289H596Calu-3H520SK-MES 1A549H1395H4600.11101001000200040006000RkoHCT116Colo 320HT-29WiDrColo206fSW480HCT15GeoRelative miR-34a expressionRelative miR-34a expression (Log scale)Relative miR-34a expressionMDA- MB-231MCF-7b)NSCLCBRCCRC25060.11101001000RkoHCT116SW480Colo 320HT-29HCT15WiDrGeoColo 206f200Relative miR-199a expression150Relative miR-199a expression4Relative miR-199a expression(Log scale)100250H1299Calu-1H522H596Calu-3H520A549H460MDA- MB-231LXF289H1395MCF-7SK-MES-1miR-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 5NSCLCBRCCRCa)501001502002503000.11101001000MCF-7MDA- MB-2311000040080012001600RkoHCT116Colo 320HT-29WiDrColo206fSW480HCT15GeoRelative Axl mRNA expressionRelative Axl mRNA expression(Log scale)Relative Axl mRNA expressionH1299Calu-1H522H596Calu-3H520A549H1395H460LXF289SK-MES 1b)NSCLCBRCCRC0.00.40.81.2H1299Calu-1H522LXF289H596Calu-3H520SK-MES 1A549H1395H460123MCF-7MDA- MB-23220.127.116.11Axl Protein ratio: (Axl/Actin)Axl Protein ratio: (Axl/Actin)Axl Protein ratio: (Axl/Actin)0.40.0RkoSW480HT-29WiDrGeoHCT15Colo 320HCT116Colo206fc)Axlβ-Actin130 kDa43 kDa1)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 206fAxl 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 6a) Axl-protein vs invasionb) miR-34a-expression vs invasionc) miR-34a-expression vs Axl-proteinAxl protein expressionInvasive abilityR= 0.41; p=0.03Log[miR-34a expression]Axl protein expressionR= -0.81; p<0.01Log[miR-34a expression]Invasive abilityR= -0.75; p<0.01Correlations 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 7a)b)H1299Rko141412121010**8*86Percent of cells migrated6Percent of cells migrated44*22Control-miRmiR-34amiR-199aControl-miRmiR-34amiR-199amiR-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)
8 Supplementary Figure 8Axl (NM_ ): Chromosome 19miR-34a (MI000268) : Chromosome 1miR-199a1-5p (MI ): Chromosome 19miR-199a2-5p (MI ): Chromosome 1miR-199b-5p (MI ) : Chromosome 9Methylation 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