Fig 2: Stimulation of β2‐AR signaling promotes, whereas knockdown of β2‐AR suppresses, Shh‐Gli1 signaling activity in vivo and in vitro. A, Gli1, Shh and Ptch1 expression levels were detected by Western blotting and quantification of PC‐3 and LNCaP cells treated with DMSO or 1 μM ISO for 24 hours. B, Gli1, Shh, and Ptch1 expression levels were detected by Western blotting and quantification in PC‐3 and LNCaP cells transduced with a negative control shRNA (shNC) and shRNA targeting β2‐AR (shβ2‐AR) treated with DMSO or 1 μM ISO for 24 hours. C and D, Representative immunohistochemistry images and quantification of Gli1, Shh, and Ptch1 expression levels in xenografts. Scale bar, 100 μm. E, Western blotting assay of the expression of downstream genes of Shh signaling and quantification in PC‐3 and LNCaP cells stably expressing shNC or shGli1 following treatment with DMSO or 1 μM ISO for 24 hours. β2‐AR, β2‐adrenergic receptor; DMSO, dimethyl sulfoxide; ISO, isoproterenol; Ptch1, patched1; SD, standard deviation; shRNA, small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: Shh‐Gli1 signaling is required for ISO‐mediated proliferation of PCa cells. A, Colony formation assays and quantification of cells treated with DMSO, GANT61 (10 μM), or ISO (1 μM) in the absence or presence of GANT61 (10 μM). Data represent the mean ± SD of three experiments. *P < .05. B and C, Western blotting and relative quantification of mRNA expression of Gli1 in PC‐3 and LNCaP cells transduced with a control shRNA (shNC) and shRNA targeting Gli1 (shGli1). D, Colony formation assays and quantification of transduced cells targeting Gli1 without or with 1 μM ISO treatment. β2‐AR, β2‐adrenergic receptor; DMSO, dimethyl sulfoxide; ISO, isoproterenol; mRNA, messenger RNA; SD, standard deviation; shRNA, small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Lentiviral vectors were used to overexpress or silencing GLI1. Western blot analysis of GLI1 protein expression in (A) MGC-803 cells, and (C) AGS cells transfected with control shRNA (shCtl) or different GLI1 targeting sequences (shGLI1#1, shGLI1#2, shGLI1#3). Western blot showing GLI1 protein expression following overexpression of GLI1 (OE-GLI1) or the empty vector (OE-CON) transfection in (B) MGC-803 cells, and (D) AGS cells. Expression levels of GLI1 mRNA in (E and G) shGLI1 transduced and untransduced cells, and (F and H) OE-GLI1 transduced and untransduced cells. GAPDH served as a loading control. (n = 3, *P < 0.05, **P < 0.01).

Fig 5: 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).