Fig 2: LGALS1 promotes GLI1 expression in vitro. (A and C) Western blotting indicating that OE- LGALS1 efficiently induced GLI1 protein expression compared with wild-type control and negative control-transduced cells. (B and D) Transfection with shLGALS1#3 inhibited GLI1 protein expression compared with wild-type control and negative control-transduced cells. These results were confirmed at the mRNA level by qRT-PCR (E–H). GAPDH served as a loading control. (All n = 3, *P < 0.05, **P < 0.01).

Fig 3: LGALS1 enhances GLI1 expression in vivo. (A) GLI1 and GAL-1 IHC scores were positively correlated in subcutaneous GC tissues from all groups (P < 0.05; ×200 magnification). (B) IHC scores indicating that GLI1 expression was elevated in the OE-LGALS1 group (P < 0.01). (C) IHC scores indicating that GLI1 expression was abrogated in the shLGALS1#3 group (P < 0.01). LGALS1 and GLI1 mRNA expression levels in (D) OE LGALS1-transduced and untransduced cells, and (E) shLGALS1#3-transduced and untransduced cells.

Fig 4: 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 5: 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).