Fig. 3: Transcriptional profiling after LETR1-ASOKD indicates potential functions in cell growth, cell cycle progression, and migration of LECs.

a Schematic representation of the ASO-mediated perturbation strategy of two replicates of neonatal LECs and BECs derived from the same donor. Only samples with ASOKD efficiency > 50% were subjected to CAGE-Seq. b Three-dimensional scatter plot showing log2FC values calculated between single ASO against LETR1 and scrambled control ASO using EdgeR³⁴. Orange and purple dots: significantly (FDR < 0.05) up- and downregulated genes (|log2FC| > 0.5) after applying a generalized linear model (GLM) design; green dot: LETR1; orange and purple text boxes: examples of DE genes with important functions in vascular biology. c Top significantly (P value < 0.05) enriched Gene Ontology (GO) terms for biological processes of selected up- and downregulated genes after LETR1-ASOKD, using g:ProfileR³⁶ (relative depth 2–5). Terms were manually ordered according to their related biological meaning. Enriched GO terms are listed in Supplementary Data 5. d Network of significantly (FDR < 0.05) enriched biological processes in LETR1-ASOKD data after Gene Set Enrichment Analysis (GSEA)⁴⁵ generated using Cytoscape and Enrichment Map¹⁰³. Orange and purple nodes: up- and downregulated GO terms; node size: total number of genes included in the gene sets; color code of node borders: FDR values on a scale from 0.05 (white) to 0.001 (red); edge width: portion of shared genes between gene sets starting from 50% similarity. To improve visualization, related biological processes were grouped manually using Wordcloud. Enriched GO terms are listed in Supplementary Data 6. e, f Motif Activity Response Analysis (MARA) network of up- e and downregulated f transcription factor binding motifs and their connection with 255 DE genes after LETR1-ASOKD. Only genes with at least a connection are displayed. Cyan/dark yellow ellipses: differentially motifs with P value between 0.05 and 0.01; blue/brown ellipses: differentially motifs with P value < 0.01; orange and purple rectangles: up- and downregulated genes, respectively; green edges: connections between deregulated genes and active motifs; red edges: connections between motifs. Enriched TF motifs are listed in Supplementary Data 7.