Fig 2: 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

Fig 3: PLTP functions as an intermediary for AURKA‐mediated P65 phosphorylation and can be targeted by GMB‐475. A) Exogenous and endogenous Co‐IP assays show that PLTP simultaneously interacts with both AURKA and P65. B) Knockdown of AURKA abolishes PLTP‐induced phosphorylation of P65. C) In vitro kinase assay demonstrating that P65 is a direct phosphorylation substrate of AURKA. D) Schematic of PLTP functional domain prediction by the SMART database and the design of domain‐specific PLTP truncation plasmids. E) Co‐IP experiments identifying the binding domains of PLTP responsible for interaction with AURKA and P65. F) Molecular docking analysis of GMB‐475 with PLTP: (a) Overall 3D view showing GMB‐475 with carbon atoms in magenta, oxygen in red, nitrogen in blue, and the protein in green cartoon representation. (b) Enlarged 3D view showing interactions between GMB‐475 and surrounding amino acid residues. Yellow dashed lines represent hydrogen bonds, red dashed lines indicate ionic bonds, and dashed purple lines indicate aromatic ring–hydrogen stacking. (c) 2D interaction map indicating hydrogen bonds (arrows) and aromatic stacking (dashed lines). G) Co‐IP assays demonstrate that GMB‐475 disrupts the interaction of PLTP with both AURKA and P65. H) Representative tumor images from different treatment groups: Hepa1‐6‐NC + PBS; Hepa1‐6‐PLTP + PBS; Hepa1‐6‐PLTP + GMB‐475. I, J) Tumor weight (I) and growth rate (J) comparisons across treatment groups. K) Representative IHC images of tumor sections showing reduced M2 macrophage infiltration (CD163+, CD206+) and enhanced CD8+ T‐cell infiltration following GMB‐475 treatment. Scale bar: 50 µm (magnification ×200). L) Quantification of CD8+ cells by IHC in subcutaneous tumor tissues from C57BL/6J mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: PLTP promotes the infiltration and polarization of M2 macrophages by activating HCC NF‐κB signal pathway. A) Bubble plot showing KEGG pathway enrichment of genes upregulated by PLTP overexpression. B) Western blot analysis of PLTP, phospho‐P65 (p‐P65), phospho‐IκBα (p‐IκBα), and phospho‐P105 (p‐P105) in HCC cells following PLTP overexpression or knockdown. C) qRT‐PCR analysis of IL‐6, IL‐8, and CSF‐1 mRNA expression following PLTP overexpression. D) The NF‐κB pathway inhibitor triptolide suppresses PLTP‐induced activation of P65 in PLC/PRF/5 and Hep3B cells. E) Transwell migration assay showing that PLTP‐induced recruitment of THP‐1 cells is attenuated by triptolide treatment. F) Microscopic analysis revealing that triptolide blocks the morphological polarization of M0 macrophages toward the M2 phenotype induced by PLTP‐HCC conditioned medium (CM). G) qRT‐PCR analysis demonstrating upregulation of CD163 and CD206 in M0 macrophages co‐cultured with PLTP‐HCC CM, which is reversed by triptolide. H) Flow cytometry analysis showing increased numbers of CD163+ and CD206+ macrophages after co‐culture with PLTP‐HCC CM, which is inhibited by triptolide. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: PLTP binds to P65 and cooperates with AURKA to promote P65 nuclear translocation and NF‐κB activation. A) Immunofluorescence analysis showing that PLTP overexpression promotes nuclear translocation of P65. Scale bar: 50 µm. B) Western blot analysis revealing increased nuclear levels of P65 upon PLTP overexpression. C) Immunofluorescence co‐localization analysis showing subcellular co‐localization of PLTP and P65. Scale bar: 10 µm. D) Co‐immunoprecipitation (Co‐IP) assay confirming the interaction between PLTP and P65. E) Mass spectrometry analysis identifying the top five candidate proteins potentially interacting with PLTP. F) Chromatographic profile of AURKA identified by mass spectrometry. G) The 3D spatial pattern of PLTP combined with P65 and AURKA, blue representing PLTP, green representing P65 (left) and AURKA (right), and red indicating the binding regions. H) Immunofluorescence analysis showing co‐localization of PLTP and AURKA. Scale bar: 10 µm. I) Co‐IP assay confirming the interaction between PLTP and AURKA.