Fig 2: Exogenous Gal-1 promotes the proliferation and invasion of GC cells and the capacity can be reversed by NRP-1 inhibitor (EG00229). (A) CCK8 assay showed Gal-1-treated AGS and MKN-1 (group was treated with 50 ng/mL rhGal-1 for 48 hours) had stronger cell growth ability than untreated cells (n = 3). (B) Gal-1-treated AGS and MKN-1 cells (group was treated with 25 or 50 ng/mL rhGal-1 for 48 hours) showed stronger colony formation ability than untreated cells. And this effect was inhibited by treating with EG00229 (3 μM) compared with the 50 ng/mL Gal-1-treated group (n=3). (C) BrdU staining assay showing AGS and MKN-1 cells treated with Gal-1 exhibited a significantly enhanced proliferation capacity compared with the wild-type control. The proliferation capacity was abolished when the medium contained 3 μM EG00229 (n=3). Magnification: ×200. (D, E) Transwell assay and Wound-healing assay showing that AGS and MKN-1 cells treated with Gal-1 had increased cell invasion and migration abilities, and 3 μM EG00229 abolished this increase in invasive ability (n=3). Magnification: ×40.Gal-1 = galectin-1; GC = gastric cancer; NRP-1 = neuropilin-1; CCK-8 = Cell Counting Kit-8; BrdU = Bromodeoxyuridine.Statistical significance was assessed by t-test; *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Fig 3: Gal-1 attenuated the apoptotic effect of cisplatin on GC cells and the capacity can be reversed by NRP-1 inhibitor (EG00229). (A, B) TUNEL staining in AGS and MKN-1 cells after untreated, Gal-1-treated alone (treated with 50 ng/mL rhGal-1 for 48 hours), and the combination of Gal-1 and EG00229 treated with 50 ng/mL rhGal-1 for 48 hours and 3 μM EG00229 for 12 hours)were exposed to cisplatin for 48 hours. Magnification: ×200. (C, D) Western blot showing that Gal-1-treated AGS and MKN-1 cells efficiently increased the expression of anti-apoptosis proteins Bcl-2 compared with untreated group in the presence of cisplatin for 48 hours. Western blot showing that Gal-1-treated AGS and MKN-1 cells efficiently decreased the expression of apoptosis proteins Caspase-3 compared with untreated group in the presence of cisplatin for 48 hours. And the capacity can be reversed by EG00229. (E, F) Gal-1 induced MKN-1 to form subcutaneous xenograft tumors with larger volumes with cisplatin. EG00229 could inhibit this effect.Gal-1 = galectin-1; GC = gastric cancer; NRP-1 = neuropilin-1.

Fig 4: Gal-1 promotes GC cell metastasis in vivo through the NRP-1/c-JUN/Wee1 signaling pathway. (A) The enrichment of KEGG pathways and GO terms among differentially expressed genes. The DEGs was determined by GC patients divided by LGALS1 expression into high and low group using expression data from TCGA (n=375). (B) The GC patients correlation between the mRNA expression of LGALS1 and c-JUN in GEPIA. The GC patients correlation between the mRNA expression of c-JUN and Wee1 in GEPIA. (C) Relative luciferase reporter assays in HEK293 cells co-transfected with plasmid constructs containing the Wee1 promoter with a c-JUN-overexpressing construct or control construct. The result confirmed the correlation between c-JUN and Wee1. (D) Western blot showing that Gal-1 efficiently increased the expression of NRP-1, c-JUN, and Wee1 in AGS and MKN-1 cells compared with untreated cells. (E) Western blot showing NRP-1 inhibitor (3 μM EG00229) efficiently decreased the levels of protein NRP-1, c-JUN, and Wee1 in AGS and MKN-1 cells. (F) Immunofluorescence staining showing Gal-1 efficiently increased the protein expression of NRP-1, c-JUN, and Wee1 in AGS and MKN-1 cells compared with untreated cells. And this effect was inhibited by treating with EG00229 (3 μM).Data are presented as the means ± standard error of the mean.Gal-1 = galectin-1; GC = gastric cancer; NRP-1 = neuropilin-1; KEGG = Kyoto Encyclopedia of Genes and Genomes; GO = Gene Ontology; DEGs = differentially expressed genes; TCGA = The Cancer Genome Atlas; GEPIA = Gene Expression Profiling Interactive Analysis.*P<0.05, **P< 0.01, ***P<0.005, ****P<0.001.

Fig 5: Gal-1 reduced the sensitivity of GC cells to cisplatin and the capacity can be reversed by NRP-1 inhibitor (EG00229). (A, B) The IC50 value of control, Gal-1-treated alone (treated with 50 ng/mL rhGal-1 for 48 hours), and the combination of Gal-1 and EG00229 treated with 50 ng/mL rhGal-1 for 48 hours and 3 μM EG00229 for 12 hours) in AGS and MKN-1 cells were measured after treated with cisplatin for 48 hours. (C, D) BrdU staining was performed control, Gal-1-treated alone (treated with 50 ng/mL rhGal-1 for 48 hours), and the combination of Gal-1 and EG00229 (treated with 50 ng/mL rhGal-1 for 48 hours and 3 μM EG00229 for 12 hours) in AGS and MKN-1 cells were exposed to cisplatin (AGS, 40 μM; MKN-1, 20 μM) for 24 hours. Magnification: ×200. (E) Control, Gal-1-treated alone (treated with 50 ng/mL rhGal-1 for 48 hours, and the combination of Gal-1 and EG00229 treated with 50 ng/mL rhGal-1 for 48 hours and 3 μM EG00229 for 12 hours) in AGS and MKN-1 cells were exposed to cisplatin (AGS, 40 μM; MKN-1, 20 μM) for 24 hours. The cells were trypsinized and collected. One thousand cells from each treatment group were reseeded in 6-well plates. A clonogenic assay was performed. (F-H) The migration and invasion of AGS and MKN-1 cells after untreated, Gal-1-treated alone (treated with 50 ng/mL rhGal-1 for 48 hours, and the combination of Gal-1 and EG00229 treated with 50 ng/mL rhGal-1 for 48 hours and 3 μM EG00229 for 12 hours)were exposed to cisplatin (AGS, 40 μM; MKN-1, 20 μM) for 48 hours. Magnification: ×40.Data are presented as the means ± standard error of the mean.Gal-1 = galectin-1; GC = gastric cancer; NRP-1 = neuropilin-1; IC50 = half maximal inhibitory concentration; BrdU = Bromodeoxyuridine.*P<0.05, **P<0.01, ***P<0.005, ****P<0.001.