Fig 1: RNA-seq validation. Bar graphs showing relative expression of AMIGO2 in SVPs after mechanical stimulation assessed by real-time qPCR. (A) Analysis performed in the same samples of the RNA-Seq (N = 5); difference among groups are evaluated using one-way ANOVA for normal distributed values, based on the result of normality tests. (B) Analysis performed using SVPs obtained from a new set of donors (N = 7), statistical analysis conducted using Kruskal–Wallis test for not normal distributed values, based on results from normality tests. Data are presented as mean ± SEM of 2-ddCt vs static (Stat) condition of each time point, *p < 0.05 **p < 0.01 (t-test stat vs. dyn).
Fig 2: AMIGO2 expression in vein graft remodeling. (A) Confocal microscopy analysis of paraffin-embedded porcine SVs for AMIGO2 (red) expression, nuclei are labeled with DAPI. Scale bar indicates 20 μm. (B) Bar graphs showing the percentage of AMIGO2 positive cells in porcine SV adventitia after grafting into the carotid artery (T0: N = 3, Day 1: N = 3, Day 7: N = 5, Day 14: N = 4, Day 90: N = 5; all data shown as Mean ± SEM, * = p < 0.05). (C) Confocal microscopy analysis of paraffin-embedded human SVs for CD34 (green), AMIGO2 (red), CD31 (white), nuclei are labeled with DAPI. Scale bar indicates 50 μm.
Fig 3: AMIGO2 upstream regulators-gene interaction networks. (A) Network depicting AMIGO2 upstream regulators predicted activity at 24 h (24 h dyn vs. 24 h stat). (B) Network depicting AMIGO2 upstream regulators predicted activity at 72 h (72 h dyn vs. 72 h stat). The values underneath each gene (when expressed in the dataset) indicate respectively the logFC and the adjusted p-value from the DE analysis. AMIGO2 is circled in red.
Fig 4: AMIGO2 associated functions. (A) Bubbleplot portraying a selection of “Diseases and Biological Functions” enriched pathways involving AMIGO2 from IPA analysis, in the 24 h dyn vs. 24 h stat comparison. (B) Bubbleplot portraying a selection of “Diseases and Biological Functions” enriched pathways involving AMIGO2 from IPA analysis, in the 72 h dyn vs. 72 h stat comparison. Pathways are organized by macro-categories on the x-axis and ordered by -log10BH-adjusted p-value on the y-axis. The bubble size is proportional to the number of molecules involved in the pathway.
Fig 5: Transcription factors potentially regulating AMIGO2. Hierarchically clustered heatmap showing differentially expressed TFs, that are predicted to regulate AMIGO2. The motif-based TF prediction was performed using iRegulon on the complete set of DEGs at both time points.
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