Fig 2: Elevated GOLPH3 expression enhances the migration/invasion abilities and stem cell-like phenotype of NSCLC cells in vitro.a GSEA indicates that GOLPH3 expression is positively associated with metastasis and stemness signatures (RICKMAN_METASTASTS_UP; RAMALH_ STEMNESS_UP). b Western blot analysis of GOLPH3 expression in the indicated cell lines stably expressing GOLPH3. c Migration assay of the indicated cell lines stably expressing GOLPH3 (left). The ratio of cells migrated across the Transwell membrane (right). d Invasion assay of cell lines stably expressing GOLPH3 (left). The ratio of cells invading the Matrigel in Transwell chambers (right). e Representative micrographs of tumor-sphere formation by GOLPH3-overexpressing or vector-transduced cells (left). Number of tumor spheres formed by the indicated cells (right). f Representative micrographs of tumor-sphere formation by GOLPH3-silenced or control cells (left). Number of tumor spheres formed by the indicated cells (right). g Flow cytometric analysis of the proportion of SP+ NSCLC cells. Data represent the mean ± SD of three independent experiments; *P < 0.05.

Fig 3: Exosomes derived from GOLPH3-overexpressing cells regulate NSCLC metastasis and a stem cell-like phenotype via activation of the WNT/ß-catenin signaling pathway.a GSEA of publicly available NSCLC profiles (REACTOME_SIGNALING_BY_WNT) suggests that GOLPH3 mRNA expression is associated positively with the activation of WNT signaling. b Luciferase-reporter assays of TOP/FOP transcriptional activity in the indicated NSCLC cells. c GO analysis of differentially expressed genes identified by IP-MASS analysis of SFB-vector and SFB-GOLPH3 cells. d Electron micrograph of exosomes isolated from GOLPH3-overexpressing and vector control cells (left; scale bar, 200 nm). Western blot analysis of CD9 and CD81 levels in exosomes of GOLPH3-overexpressing and vector control cells (right). e, f Migration assay of the GOLPH3-silenced cell line treated with exosomes from A549-GOLPH3 cells (e) or NCI-H460-GOLPH3 cells (f) (left). The number of cells that migrated to the lower Transwell chamber, *P < 0.05, right. g, h Migration assay of A549-GOLPH3 cells (g) and NCI-H460-GOLPH3 cells (h) treated with GW4869 (left). Number of cells that migrated to the lower Transwell chamber; *P < 0.05, right. i Real-time PCR analysis of the mRNA expression of metastasis-related markers in GOLPH3-overexpressing and vector cells treated with GW4869.

Fig 4: GOLPH3 interacts with CKAP4 to promote NSCLC metastasis and a cancer stem cell-like phenotype.a IP-MASS analysis showing GOLPH3-binding proteins. CKAP4 is the top differentially interacting protein in comparisons of SFB-vector and SFB-GOLPH3 cells by IP-MASS analysis; exosome-related proteins are marked in pink; the others are marked in blue. b Co-IP analysis of the interactions between GOLPH3 and CKAP4 in A549 and NCI-H460 cells. c Co-IP analysis of the interaction between GOLPH3 and CKAP4 in A549 and NCI-H460 cells transfected with SFB-Vector and SFB-GOLPH3. d, e Proximity ligation assay (PLA) of A549 (d) and NCI-H460 (e) cells with anti-GOLPH3 (Mx) and anti-CKAP4 (Rx) antibodies. Representative image of PLA signals (left); statistical analysis (right). Data represent the mean ± SEM of three independent experiments; ****P < 0.0001, one-way ANOVA. f Migration assay of GOLPH3-overexpressing cell lines treated with siCKAP4 or siRNA-vector (left). The number of cells that migrated to the lower Transwell chamber (right); *P < 0.05. g Real-time PCR analysis of the mRNA expression of metastasis-related or stemness-related proteins in GOLPH3-overexpressing and vector control cells with siCKAP4 or siRNA-vector treatment, respectively.

Fig 5: GOLPH3 overexpression enhances the tumorigenicity and metastasis of NSCLC cells in vivo.a Tumors formed by different numbers of A549 cells (1 × 106, 1 × 105, 1 × 104, or 1 × 103) transduced with pMSCV-GOLPH3, pMSCV-vector, pSuper-GOLPH3-shRNA#1, or pSuper-vector plasmid. b Tumor growth curves following implantation of 1 × 106, 1 × 105, 1 × 104, or 1 × 103 cells. c–e Histological confirmation of tumor cells by H&E staining. Representative images of an IHC assay of MUC1 in metastatic lesions in the intestine (c), diaphragm (d), and liver (e). f Histological confirmation of tumor cells by H&E staining. Representative images of an IHC assay of GOLPH3, CD133, and ZEB1 in subcutaneous tumors formed by the indicated cells.