Fig 2: (A,B) Immunostaining showing the subcellular localization of ß3GalT-IV and ST3Gal-II, respectively. and its colocalization with GM130 (130 kDa cis-Golgi matrix protein). Scale bars: 10 µm. (C,D) Quantification of the colocalization between GM130 with ß3GalT-IV or ST3Gal-II, respectively. Two-tailed, unpaired t tests were carried out to assess statistical significance of results (ns: not significant). (E) Homogenates from T98G control and GOLPH3 knockdown (KD) cells transiently expressing a full-length, HA-tagged version of ST3Gal-II were treated (+) or not (-) with endoglycosidase H and subjected to SDS-PAGE and Western blot. As control, CHO-K1 cells were included in the analysis. CHO-K1 cells were transfected to express the wild-type, full-length, HA-tagged version of ST3Gal-II in the presence (+) or absence (-) of tunicamycin or an N-glycosylation mutant variant of the enzyme (ST3Gal-II N211Q). The expected molecular weight of unglycosylated and N-glycosylated ST3Gal-II is 41 and 43 kDa, respectively.

Fig 3: WNT3A in exosomes derived from GOLPH3-overexpressing cells enhances NSCLC cell metastasis and a stem cell-like phenotype.a Western blot analysis of WNT3A levels in exosomes derived from GOLPH3-overexpressing and vector control cells (left); quantification of WNT3A expression (right). b ELISA of WNT3A levels in GOLPH3-overexpressing, GOLPH3-silenced, or vector control cells. c ELISA of WNT3A levels in exosomes derived from GOLPH3-overexpressing and vector control cells. d Western blot analysis of WNT3A levels in exosomes derived from GOLPH3-overexpressing cells treated with GW4869 (left); quantification of WNT3A expression (right). e ELISA of WNT3A levels in GOLPH3-overexpressing cells treated with GW4869. f ELISA of WNT3A levels in exosomes of GOLPH3-overexpressing cells treated with GW4869 inhibitors. g, h Real-time PCR analysis of the mRNA expression stemness-related markers (g) and metastasis-related markers (h) in GOLPH3-silenced and vector control cells treated with exosomes or recombinant WNT3A protein.

Fig 4: GOLPH3 overexpression promotes distal metastasis of NSCLC xenografts in vivo.a For the intravenous (tail vein) injection metastasis model, bioluminescent images of subcutaneous tumors generated from the indicated cells and distant metastasis signals are shown. b–d Histological confirmation of tumor cells by H&E staining. Representative images of an IHC assay of the lung adenocarcinoma marker (MUC1) in metastatic lesions in the lung (b), intestine (c), and brain (d). e For the intracardiac injection transfer model, bioluminescent images of subcutaneous tumors of A549-luc-vector, A549-luc-GOLPH3, A549-luc-RNAi-vector, and A549-luc-GOLPH3-sh2; distant metastasis signals are also shown. f–h Histological confirmation of tumor cells by H&E staining. Representative images of an IHC assay of MUC1 in metastatic lesions in the lung (f), intestine (g), and brain (h).

Fig 5: 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.