Fig 1: SENP1 regulates LR-MSC myofibroblast transition through both the WNT/β-Catenin and Hedgehog/GLI signaling pathways in vitro. A mRNA expression of endogenous SENP1, β-Catenin, and GLI1 were assessed using RT-PCR in different groups. RT-PCR analysis indicating that β-Catenin and GLI1 mRNA levels did not change in the presence of LV-SENP1-shRNA during TGFβ1 induction. *P < 0.01, **P < 0.05, ▲P < 0.01, ▲▲P > 0.05. *, the control group vs. the TGFβ1 group; **, the SENP1 knockdown group vs. the TGFβ1 group; ▲, the control group vs. the TGFβ1 group; ▲▲, the SENP1 knockdown group vs. the TGFβ1 group. B SENP1 level in total cell lysates was assessed using western blot analyses. *P < 0.01, **P < 0.05. *, the control group vs. the TGFβ1 group; **, the SENP1 knockdown group vs. the TGFβ1 group. C β-Catenin and GLI1 levels in total cell lysates were assessed using western blot analyses. The SUMOylation analysis of the immunoprecipitated β-Catenin and GLI1 proteins. β-Catenin and GLI1 were immunoprecipitated from whole-cell lysates using anti-β-Catenin and anti-GLI1 antibodies, respectively. The blots were probed with SUMO1. *P < 0.01, **P < 0.05, ▲P < 0.01, ▲▲P < 0.05. *, the control group vs. the TGFβ1 group; **, the SENP1 knockdown group vs. the TGFβ1 group (β-Catenin and GLI1); ▲, the control group vs. the TGFβ1 group; ▲▲, the SENP1 knockdown group vs. the TGFβ1 group (β-Catenin-SUMO and GLI1-SUMO). All data are presented as the mean ± SEM of three independent experiments. Quantification is shown in the lower panel. Each experiment was performed in triplicate.
Fig 2: miR-367-3p overexpression downregulates the expression of Rab23 and inhibits the Hedgehog signaling pathway. (A) Detection of Gli1 expression in DU145 cells transfected with the miR-367-3p mimic and inhibitor. (B) Detection of Gli1 expression in PC3 cells transfected with the miR-367-3p mimic and inhibitor. (C) Detection of Gli2 expression in DU145 cells transfected with the miR-367-3p mimic and inhibitor. (D) Detection of Gli2 expression in PC3 cells transfected with the miR-367-3p mimic and inhibitor. (E) Gli1 and Gli2 expression levels were detected by western blot analysis in DU145 cell lines. *P<0.05, **P<0.01, ***P<0.001.
Fig 3: Downregulation of GLI1 attenuates glucose metabolism in MDA-MB-231 cells. (A) Glycolytic rate of MDA-MB-231 cells after siRNA transfection was measured using the XF Glycolytic Rate Assay Kit. (B) Both basal and compensatory glycolysis decreased after GLI1 knockdown. (C) Quantitative levels of lactate in cell media. GLI1 siRNA significantly reduced the lactate levels in the media of MDA-MB-231 cells. **p < 0.01 as compared with the control siRNA-transfected cells. (D) Expression patterns of glucose metabolism-related proteins after GLI1 knockdown by siRNA. qRT-PCR analysis of key glycolytic enzymes in siRNA-transfected MDA-MB-231 cells. (E) Western blot analysis of glucose metabolism-related proteins in MDA-MB-231 cells. (F) Intensities of the bands were measured and depicted in the bar graph as the ratio of the expression of the protein to that of β-actin. *p < 0.05, **p < 0.01 compared with the control siRNA-transfected cells.
Fig 4: 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 5: GLI1 promotes VM in vivo and in vitro. (A) Matrigel three-dimensional culture showing that GLI1 overexpression enhanced tube formation in MGC-803 and AGS cells, while silencing of GLI1 in MGC-803 and AGS cells inhibited their ability to form tube-like structures (×40 magnification; n = 3). (B) Expression of GLI1 and the formation of VM structures in the OE-GLI1 and sh-GLI1 subcutaneous GC mouse groups detected by IHC and CD34/PAS double staining. VM was significantly increased in the OE-GLI1 group, and was absent in the shGLI1#3 group (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).
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