Fig. 7
EV-packaged circTLCD4-RWDD3 promotes PROX1 expression in HLECs. a qRT-PCR analysis of lymphangiogenesis-related gene expression in A549-EVVector- or A549-EVcircTLCD4-RWDD3-treated HLECs. b Western blotting analysis to verify PROX1 expression in HLECs treated with A549-EVsi-NC, A549-EVsi-circTLCD4-RWDD3#1, or A549-EVsi-circTLCD4-RWDD3#2. c Western blotting analysis to verify PROX1 expression in HLECs treated with A549-EVVector or A549-EVcircTLCD4-RWDD3. d Transcriptional activity of PROX1 in A549-EVcircTLCD4-RWDD3-treated HLECs transfected with truncated PROX1 promoter luciferase plasmids. e ChIRP assays to investigate the circTLCD4-RWDD3-associated chromatin fragments of the PROX1 promoter in HLECs. f Schematic representation of the DNA-RNA triplex structure between circTLCD4-RWDD3 and the PROX1 promoter. Schematic was created with BioRender (www.biorender.com). g Luciferase activity detected in A549-EVVector- or A549-EVcircTLCD4-RWDD3-treated HLECs with or without mutating the circTLCD4-RWDD3-binding site on PROX1 promoter. h, i ChIP-qPCR of the enrichment of hnRNPA2B1 (h) and H3K4me3 (i) on PROX1 promoter in A549-EVVector- or A549-EVcircTLCD4-RWDD3-treated HLECs. j–l Representative images (j) and quantification of Transwell migration (k) and tube formation (l) of A549-EVVector- or A549-EVcircTLCD4-RWDD3-treated circTLCD4-RWDD3KO HLECs with or without knocking down PROX1. Scale bars, 100 µm. The statistical difference was assessed by the unpaired Student’s t-test in (a, d, e, h, and i); and one-way ANOVA followed by Dunnett tests in (g, k, and l). Error bars show the SD from three independent experiments. *P < 0.05; **P < 0.01
