Fig. 8: LETR1 interacts with several protein complexes to exert its transcriptional regulatory function. | Nature Communications

Fig. 8: LETR1 interacts with several protein complexes to exert its transcriptional regulatory function.

From: LETR1 is a lymphatic endothelial-specific lncRNA governing cell proliferation and migration through KLF4 and SEMA3C

Fig. 8

a Protein–protein interaction network using Search Tool for Recurring Instances of Neighbouring Genes (STRING)66 of the 59 protein targets after in vitro biotin-RNA pull-down followed by mass spectrometry of LETR1 and its antisense control in LECs (n = 2). Proteins were clustered by Markov Clustering (MCL) algorithm124 (inflation parameter = 1.5). Circles: most relevant clusters; lines: interactions within each complex; thickness of the lines: interaction confidence from text mining, databases, experiments, and co-expression. Disconnected nodes were hidden to improve visualization. Interacting proteins are listed in Supplementary Data 12. b Graph showing log2FC LECs versus BECs against average unique peptide after LETR1 RNA pull-down in LECs (n = 2). Blue lines: log2FC > 0.5 and unique peptide > 5 thresholds. c Representative western blot after RBBP7 RNA Immunoprecipitation (RIP) followed by qPCR for LETR1 in LECs. To prevent IgG heavy chain masking, a conformation-specific IgG secondary antibody was used. d Enrichment of LETR1 displayed as FC against IgG control after RBBP7 RIP in LECs. e Expression levels of KLF4 and SEMA3C after LETR1-ASO2 knockdown in LEC Chromatin Immunoprecipitation (ChIP) samples. f Representative western blot images after LETR1-ASO2 knockdown followed by ChIP-qPCR for RBBP7, RNA Pol II, H3K4me3, and H3K27me3 in LECs. g–j Enrichment for RBBP7 (g), RNA Pol II (h), H3K4me3 (i), and H3K27me3 (j) at KLF4 and SEMA3C genomic loci displayed as FC against IgG control. UNTR: negative control for RBBP7 and RNA Pol II; GAPDH: positive control for RNA Pol II and H3K4me3 and negative control for H3K27me3; MYT: positive or negative control for H3K27me3 and H3K4me3. k Model for the mode of action of LETR1 in regulating cell growth and cell migration in LECs. Data are displayed as mean values + SD (n = 3 in d, e, and g–j). Percentages represent LETR1 knockdown efficiencies after the experiments. *P < 0.05, ns: not significant, n.d.: not detected using unpaired two-tailed Student’s t test against IgG control (d), scrambled ASO control (e), and ChIP target—LETR1-ASO2 (g–j). All displayed experiments were performed in neonatal LECs derived from the same donor. Uncropped western blot images are shown in Supplementary Figure 9.

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