Fig 1: LINC01614 enhances ANXA2 and p65 interactions and promotes NF-?B 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-?B target genes in the exosome-packaged LINC01614 treated A549 cells. J NF-?B activity of CAF-exosome-treated A549 cells, examined by luciferase reporter assay (n = 3). K Western blot analysis of the nuclear factor NF-?B 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-?B activity of A549 transduced with lenti-LINC01614, determined by luciferase reporter assay (n = 3). N Western blot analysis of the nuclear factor NF-?B 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
Fig 2: The expression of ANXA2 in MSCs during osteogenic differentiation under high-glucose condition. Western blot analysis demonstrated ANXA2 expression at day 7 (a) and day 14 (b) in MSCs in growth medium (CTRL), ODM (5.5 mM glucose), ODM with 10 mM (10G), 25 mM (25G), and 40 mM (40G) glucose. The protein band intensity is presented as relative to CTRL. All data are presented as the means ± SEM from three independent experiments. * p value < 0.05 is considered statistically significant.
Fig 3: LINC01614 promotes ANXA2-dependent p65 phosphorylation and the transcription of SLC38A2 and SLC7A5. A, D Exosomes isolated from the CM of CAFs transduced with lenti-LINC01614-shRNA were added into A549 cells for 48 h. Exosomes from shctrl CAFs-treated A549 cells were used as controls. A, B Western blotting for total and phosphorylated IKK and I?Ba in A549 cells with indicated treatments. C A549 cells were transduced without or with lenti-LINC01614 and pretreated with an inhibitor of NF-?B nuclear translocation (JSH-23) or IKK inhibitor (BAY 11-7-82). Immunofluorescent p65 staining showing its nuclear translocation in A549 cells (n = 3). D Expression of ANXA2, total, Ser276, and Ser536 phosphorylation of p65 in A549 cells with indicated treatments (n = 3). E Overexpressing LINC01614 or ANXA2 promoted Ser276 phosphorylation of p65 in A549 cells (n = 3). F Knockdown of ANXA2 abrogated the effects of LINC01614 on Ser276 phosphorylation (n = 3). G Representative immunofluorescent images of p65 nuclear translocation in A549 cells with indicated treatments. Scale bars, 50 µm. H NF-?B activity of A549 cells with indicated treatments. I qRT-PCR analysis of SLC38A2 and SLC7A5 in A549 cells with indicated treatments. J A conserved NF-?B binding element at the promoters of SLC38A2 and SLC7A5 were predicated by JASPAR (n = 3). K ChIP-PCR analysis for NF-?B occupancy at the promoters of SLC38A2 and SLC7A5 in A549 cells (n = 3). L Luciferase reporter assays of the transduced A549 cells transfected with reporter plasmids containing the SLC38A2 and SLC7A5 promoter, respectively. Wild type: -2000–0 construct; mutant: -2000–0 constructed with a point mutation at the NF-?B binding site. Transduced A549 cells transfected with a blank pGL3 plasmid used as a negative control (n = 3). M Graphic for ENCODE database of p65 ChIP-seq. For H, I and K, L, 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; IP immunoprecipitation; LUAD lung adenocarcinoma
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