Fig. 4: Inhibition of GLI1 induces apoptosis of MF fibrocytes.

A Representative micrographs of MF BM LDC-derived fibrocytes transfected with GLI1-siRNA or Ctrl-siRNA were analyzed by fluorescence immunostaining. Fibrocytes were cultured from BM LDCs of MF patients and transfected with GLI1-siRNA for 48 h. Cells were stained for GLI1 (Alexa Fluor 594, pseudo-colored red) and F-actin (Alexa Fluor 488, pseudo-colored green) with DAPI (pseudo-colored blue) as a nuclear counterstain. B Quantitation of fibrocytes from MF patients (n = 4) following GLI1 silencing showed that GLI1-siRNA induced a significant reduction in fibrocyte counts. Fibrocyte counts were assessed by using an unsupervised cell segmentation algorithm, and the mean cell number per unit area was calculated for each well. The paired t-test assessed the statistical significance of differences between matched samples. C Representative micrographs of GANT61-treated MF BM LDC-derived fibrocytes analyzed by fluorescence immunostaining. Cultured viable and morphologically intact fibrocytes were incubated with 10 µM GANT61 for 72 h. Cells were stained for GLI1 (Alexa Fluor 594, pseudo-colored red) and F-actin (Alexa Fluor 488, pseudo-colored green) with DAPI (pseudo-colored blue) as a nuclear counterstain. D Quantitation of fibrocytes from MF patients (n = 4) following treatment with GANT61 showed that GANT61 significantly reduced fibrocyte numbers. An unsupervised cell segmentation algorithm determined fibrocyte numbers per unit area, and the means in each well were calculated. Matched values were compared using the paired t-test. E DNA laddering analysis of GLI1-silenced fibrocytes. Fibrocytes were cultured from BM LDCs of MF patients (n = 2) and BM LDCs of an NC and transfected with GLI1-siRNA or Ctrl-siRNA. Total genomic (g) DNA was isolated and 0.5 µg of gDNA was separated using agarose gel electrophoresis. DNA fragmentation observed in GLI1-silenced MF fibrocytes suggests that GLI1-siRNA, but not Ctrl-siRNA, significantly reduced fibrocyte viability. F The panel depicts representative micrographs of fibrocyte apoptosis rates assessed by TUNEL staining. High levels of biotinylated dNTPs (Alexa Fluor 488, pseudo-colored green) were detected in MF fibrocytes transfected with GLI1-siRNA. In contrast, biotinylated dNTPs were not detected in NC fibrocytes transfected with GLI1-siRNA or in MF fibrocytes transfected with Ctrl-siRNA. In addition, cell nuclei (stained with DAPI, pseudo-colored blue) show signs of karyorrhexis not observed in NC fibrocytes or fibrocytes transfected with Ctrl-siRNA. All micrographs were adjusted for brightness and contrast linearly and equally between groups. Scale bars represent 100 µm. *P < 0.05; **P < 0.01, respectively.