Fig. 5: GLI1 activates pro-fibrotic signaling pathways in MF fibrocytes.

A, B Quantitation of GLI1, MMP2, MMP9, and procollagen-I (COL1A1) levels in MF fibrocytes following GLI1 silencing. Cultured fibrocytes from MF patients were transfected with GLI1-siRNA or Ctrl-siRNA and expression analyses were performed 48 h following siRNA transfection. As shown, GLI1 silencing induced a significant reduction in GLI1, MMP2, and MMP9 mRNA (A n = 9) and protein levels (B n = 4) as assessed by qRT-PCR and western immunoblotting, respectively. C, D Quantitation of GLI1, MMP2, MMP9, and procollagen-I (COL1A1) levels in fibrocytes treated with GANT61. Cultured fibrocytes from MF patients were incubated with 10 µM GANT61 for 72 h or left untreated (Ctrl). Neither expression analyses of mRNA by qRT-PCR (A n = 9) nor protein by western immunoblotting (B n = 5) showed significant change in the levels of the analyzed genes following treatment with GANT61. E Protein levels of GLI2, GLI1, MMP2, and MMP9 in MF fibrocytes after GLI2 silencing. Cultured BM fibrocytes from MF patients (n = 3) and NCs (n = 2) were transfected with GLI2-siRNA or Ctrl-siRNA and expression analyses were performed 48 h following siRNA transfection. As shown by western immunoblotting, GLI2 silencing had no significant effect on GLI1 or its target genes. F Protein levels of procollagen-I, phosphorylated (p) and total SMAD2 and SMAD3 in GLI1-silenced fibrocytes were analyzed by western immunoblotting. Fibrocytes from MF patients (n = 4) and an NC were transfected with GLI1-siRNA or Ctrl-siRNA and their protein was collected after 48 h. As shown, GLI1 silencing reduced procollagen-I and phosphorylated SMAD2/3 levels. The PPIA gene was used as an internal control in the qRT-PCR analyses. Matched values were compared by using the paired t-test. All target proteins were detected by sequential probing of the same blot and β-actin was used as loading control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, respectively.