Presentation on theme: "Supplementary Figure 1 b) Human miRNA-199a - Axlc) Human miRNA-199b - Axl a) Human miRNA-34a - Axl Predicted secondary structures (Pic Tar predicitions)"— Presentation transcript:
Supplementary Figure 1 b) Human miRNA-199a - Axlc) 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 ( 3UTR 24 to 50 nt) and miR-34a, Axl ( 3UTR 25 to 56 nt) and miR-199a/b. Green- sequences represent the miR; a Red-sequence represents the Axl-3UTR
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-miR2: PM-miR-34a3: PM-miR-199a H Rko
Supplementary Figure a) PM-miRNA expression Relative miR expression (Log Scale) Con-miR miR-34a miR-199a Rko Con-miR miR-34a miR-199a H Con-miR miR-34a miR-199a MDA-MB Con-miR miR-34a miR-199a HCT116 Con-miR miR-34a b) AM-miRNA expression Relative miR expression Con-miR miR-34a Con-miR miR-34a A549 MCF7Colo 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 Rko HCT116 SW480 Colo 320 HT-29 HCT15 WiDr Geo Colo 206f MDA- MB-231 MCF H1299 Calu-1 H522 LXF289 H596 Calu-3 H520 SK-MES-1 A549 H1395 H460 Relative miR-199a expression a) NSCLCBRCCRC Rko HCT116 Colo 320 HT-29 WiDr Colo206f SW480 HCT15 Geo Relative miR-34a expression H1299Calu-1 H522 LXF289 H596 Calu-3 H520 SK-MES 1 A549 H1395 H MCF-7 MDA- MB-231 b) NSCLCBRCCRC 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 Relative miR-199a expression Relative miR-34a expression (Log scale) Relative miR-199a expression(Log scale) Relative miR-34a expression
Rko HCT116 Colo 320 HT-29 WiDr Colo206f SW480 HCT15 Geo Relative Axl mRNA expression H1299Calu-1 H522 LXF289 H596 Calu-3 H520 SK-MES 1 A549 H1395 H460 Axl Protein ratio: (Axl/Actin) H1299 Calu-1 H522 LXF289 H596 Calu-3 H520 SK-MES 1 A549 H1395 H MCF-7 MDA- MB MCF-7 MDA- MB Rko HCT116 Colo 320 HT-29 WiDr Colo206f SW480 HCT15 Geo NSCLCBRCCRC a) b) NSCLCBRCCRC Supplementary Figure 5 Axl 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-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 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 Relative Axl mRNA expression(Log scale) Axl Protein ratio: (Axl/Actin) Relative Axl mRNA expression Axl Protein ratio: (Axl/Actin)
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 Axl protein expression Invasive ability R= 0.41; p=0.03 c) miR-34a-expression vs Axl-protein a) Axl-protein vs invasionb) 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 Spearmans correlation test).
Supplementary Figure 7 H Control-miRmiR-34amiR-199a Percent of cells migrated Rko Percent of cells migrated Control-miRmiR-34amiR-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 (*p 0.05)
miR-34a (MI000268) : Chromosome 1 miR-199a1-5p (MI ): Chromosome 19 Supplementary Figure 8 Axl (NM_ ): Chromosome 19 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 miR-199a2-5p (MI ): Chromosome 1