Fig. 4
LINC01614 enhances ANXA2 and p65 interactions and promotes NF-κB activation. A p65 and ANXA2 were pulled down by biotin-labeled LINC01614 but not LINC01614 antisense RNA in whole-cell lysates of A549 cells treated with exosomes from CAFs (n = 3). B, C RIP evaluation of the interaction between ANXA2 (B) and p65 (C) using anti-ANXA2 and anti-p65 antibodies in A549 cells treated with exosomes from CAFs (n = 3). D ANXA2 and LINC01614 were co-precipitated with p65 in whole-cell lysates of A549 cells treated with exosomes from CAFs (n = 3). E IP (immunoprecipitation) analysis for the in vitro interaction of ANXA2 and p65. LINC01614 promoted the binding between recombinant ANXA2 and p65 in vitro (n = 3). F The secondary structure of LINC01614 is shown as predicated by the centroid method (http://rna.tbi.univie.ac.at). RNA pull-down assay for the interactions of sequentially deleted LINC01614 variants with ANXA2 and p65 in ectopic LINC01614 expressed A549 cells (n = 3). Schematic of sequentially deleted LINC01614 variants (left). Representative western blot for ANXA2 and p65 pulled down by LINC01614 variants (right). G nucleotide mutation lacking the stem-loop structure of LINC01614 (973–1775) abolished the interaction between p65 and ANXA2 as revealed by IP analysis. H LINC01614973–1775 enhanced the interaction between p65 and ANXA2, as revealed by IP analysis. I GSEA revealed enrichment of NF-κB target genes in the exosome-packaged LINC01614 treated A549 cells. J NF-κB activity of CAF-exosome-treated A549 cells, examined by luciferase reporter assay (n = 3). K Western blot analysis of the nuclear factor NF-κB p65 subunit following nuclear fractionation of exosome treated A549 cells. L Immunofluorescent p65 staining showing nuclear translocation in A549 cells with indicated treatments (n = 3). M NF-κB activity of A549 transduced with lenti-LINC01614, determined by luciferase reporter assay (n = 3). N Western blot analysis of the nuclear factor NF-κB p65 subunit following nuclear fractionation of A549 cells transduced with LINC01614. Loading controls, GAPDH (cytoplasmic fractions), and H3 (nuclear fractions) (n = 3). O Immunofluorescent p65 staining showing its nuclear translocation in A549 cells with indicated treatments (n = 3). For B, C, J, and M means ± s.d. are shown, and independent sample t-tests determined P values. *P < 0.05, **P < 0.01, ***P < 0.001. UT cancer cells without any treatment; CM conditioned medium; Exos exosomes; GESA gene set enrichment analysis; LUAD lung adenocarcinoma