Fig. 3: GLI1 is required for fibrocyte formation in vitro.

A Representative micrographs of fibrocytes formed after exposure of BM LDCs to 0, 2.5, 5, and 10 μM GANT61 and analyzed by fluorescence cytochemistry. BM LDCs from MF patients and NCs were cultured in conditions favoring differentiation to fibrocytes and GANT61 was added 24 h following culture initiation. After 7 days cells were stained for F-actin (Alexa Fluor 488, pseudo-colored green) and their nuclei were counterstained using DAPI (pseudo-colored blue). As shown, 7-day treatment with GANT61 reduced the numbers of both MF and NC fibrocytes in a dose-dependent manner. All micrographs were adjusted for brightness and contrast linearly and equally between groups. The scale bar represents 1 mm. B Quantitation of fibrocyte formation following 7-day treatment with GANT61. Fibrocyte numbers were assessed by using an unsupervised cell segmentation algorithm and the mean cell number per unit area was calculated for each well. The significance of change at different concentrations of GANT61 was assessed using repeated-measures ANOVA followed by Dunnett’s multiple comparisons test. C Representative annexin V/PI flow cytometry plots of GLI1-silenced BM LDCs from an MF patient. BM LDCs from MF patients were transfected with GLI1-siRNA or Ctrl-siRNA for 24 h and stained using annexin V (FITC) and PI. Untreated BM LDCs from the same patient served as a negative control (Ctrl). To measure the percentage of apoptotic cells annexin V-positive cells were gated in each sample. As shown, the fraction of apoptotic cells was 50% higher in GLI1-silenced LDCs than in both controls. D Quantitation of apoptotic cell fractions after GLI1 silencing of LDCs from MF patients (n = 5). The difference between siRNAs was assessed using repeated-measures ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001, respectively.