Fig. 5

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 IκBα in A549 cells with indicated treatments. C A549 cells were transduced without or with lenti-LINC01614 and pretreated with an inhibitor of NF-κB 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-κB 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-κB binding element at the promoters of SLC38A2 and SLC7A5 were predicated by JASPAR (n = 3). K ChIP-PCR analysis for NF-κB 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-κB 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