Fig 2: Study overview. Diagram showing patients and cell lines included and the analyses performed in the study. ECAD, E-cadherin; ITGB4, integrin ß4; IVD, in vitro diagnostic; MSI, microsatellite instable; MSS, microsatellite stable; R0, complete resection/no residual tumor; TMA, tissue microarray; ZO-1, zona occludens-1.

Fig 3: SPC25 activates the FAK/PI3K/AKT signaling pathway through ITGB4. (A) Western blot analysis revealed the changed levels in phosphorylated (p)FAK, pPI3K and pAKT after SPC25 or ITGB4 silencing in Huh7 and HCCLM3 cells (*P<0.05, **P<0.01, ***P<0.001; ns, not significant, independent-samples t-test). (B) Western blot analysis, revealing the results of the rescue experiment on proteins associated with the FAK/PI3K/AKT signaling pathway (***P<0.001; ns, not significant, one-way ANOVA). (C and D) The altered levels of MMP9 and MMP13 in Huh7 and HCCLM3 after SPC25 silencing are shown [(C) Results of western blot, (D) Results of RT-qPCR (**P<0.01, ***P<0.001, independent-samples t-test]. SPC25, spindle pole body component 25 homolog; FAK, focal adhesion kinase; ITGB4, integrin subunit β4; p, phosphorylated; MMP, matrix metalloproteinase; PI3K, phosphoinositide 3-kinase.

Fig 4: Model of the signaling pathway involved in the promotion of HCC metastasis by SPC25. In the nucleus, SPC25 facilitates ITGB4 transcription. Subsequently in the cytoplasm, ITGB4 binds to laminin on the cell membrane and activates the FAK/PI3K/AKT signaling pathway, thereby promoting HCC metastasis. SPC25, spindle pole body component 25 homolog; HCC, hepatocellular carcinoma; ITGB4, integrin subunit β4; FAK, focal adhesion kinase; PI3K, phosphoinositide 3-kinase.

Fig 5: The TMEM268/ITGB4 interaction occurs at their C-terminals. a GST or GST-TMEM268 fusion protein were immobilized on glutathione-Sepharose beads and incubated with BGC823 cell lysates at 4 °C for 4 h. ITGB4 and GST were detected in the washed beads by western blotting. b Total BGC823 cell extracts were subjected to IP using either an anti-TMEM268 antibody or an IgG isotype control. ITGB4 was detected in the immunoprecipitates by western blotting. c, d Construction of truncated TMEM268 and ITGB4 plasmids. e BGC823 cells were transfected with FLAG-TMEM268, FLAG-TMEM268-N, or FLAG-TMEM268-C for 24 h. Total cell extracts were subjected to IP using either an anti-FLAG antibody or IgG isotype control. ITGB4 and FLAG were detected in the immunoprecipitates by western blotting. f GST and GST-TMEM268, GST-TMEM268-N or GST-TMEM268-C fusion protein immobilized on glutathione-Sepharose beads were incubated with BGC823 cell lysates. ITGB4 and GST were detected in the washed beads by western blotting. g 293T cells were cotransfected with FLAG-TMEM268 and either HA-ITGB4-C1, HA-ITGB4-C2 or HA-ITGB4-C3 for 24 h. Total cell extracts were subjected to IP using an anti-HA antibody. ITGB4 mutants and FLAG-TMEM268 were detected in the immunoprecipitates by western blotting