Fig 2: GAL-1 expression is correlated with GLI1, and GLI1 expression is correlated with VM. (A) Representative images of IHC indicating GLI1 and GAL-1 protein expression in GC tissues (×200 magnification). (B) The GAL-1 IHC scores in GLI1-positive GC tissues were significantly higher than the GAL-1 IHC scores in GLI1-negative GC tissues(P < 0.01) (C) GAL-1 IHC scores were positively correlated with GLI1 IHC scores in GC tissues (r = 0.958; P < 0.01). (D) GC tissues exhibiting high GLI1 expression were more likely to form VM (endogenous cell-dependent vessels are indicated by the red dotted line, VM is indicated by the green dotted line, and red blood cells are indicated by red arrows in VM and endogenous cell-dependent vessels; ×400 magnification). (E) The GLI1 IHC scores in GC tissues with VM were significantly higher than those without VM (P < 0.01).

Fig 3: GAL-1 increases VM through the Hh signaling pathway. (A) Matrigel three-dimensional culture indicating that OE-LGALS1 efficiently increased tube-formation by MGC-803 cells and AGS cells, and the inhibition of this effect by GANT61. (B) sh-LGALS1 reduces the ability of MGC-803 cells and AGS cells to form tube-like structures. Simultaneous silencing of LGALS1 and overexpression of GLI1 rescued this reduction in tube-formation capacity. (×40 magnification; n=3).

Fig 4: Galectin-1 is upregulated and required for L6 myotube formation.A, quantification of galectins during myogenesis. B, western blot analysis of galectin-1 during myogenesis. C, CRISPR/Cas9(D10A) paired gRNA strategy to KO Lgals1. D, western blot analysis of galectin-1 and 14-3-3 loading control in WT and KO L6 myoblasts. E, immunofluorescence microscopy of WT and KO cells at day 3 of differentiation (blue = Hoechst; green = myosin; white scale bar = 50 μm). F, quantification of the differentiation index expressed as the percentage of nuclei in myosin-positive cells. ∗q < 0.05 ANOVA with permutation-based FDR. #p < 0.05 two-way ANOVA. FDR, false discovery rate.

Fig 5: Lentiviral vectors were used to overexpress or silencing LGALS1 using 3 shRNAs. Western blot analysis showing decreased GAL-1 protein expression in (A) MGC-803 cells, and (C) AGS cells transfected with LGALS1 shRNAs, compared with cells transfected with control shRNA (shCtl). Western blot showing GAL-1 protein expression following overexpression of LGALS1 (OE-LGALS1) compared to the empty vector (OE-Con) in (B) MGC-803 cells, and (D) AGS cells. Reduced expression levels of LGALS1 mRNA in (E) MGC-803 cells, and (G) AGS cells transfected with LGALS1 shRNAs. Increased expression levels of LGALS1 with OE-LGALS1 in (F) MGC-803 cells, and (H) AGS cells transfected with OE-LGALS1. GAPDH was used as a loading control. (All n = 3, *P < 0.05, **P < 0.01).