Figure 1.
Constitutive GLI activity in canine osteosarcoma cell lines.
Relative transcript expression of canine osteosarcoma cell lines and canine osteoblast cells were determined by Real Time RT-PCR. (A) Abrams and D17 cell lines showed high relative expression of GLI1 compared to canine osteoblasts, whereas expression in the Moresco cell line was significantly decreased. (B) The D17 cell line showed high expression of GLI2 mRNA, whereas Moresco cells expressed a significantly lower level of transcript compared to canine osteoblast cells. (C) Abrams and D17 cell lines showed high expression of PTCH1 mRNA compared to canine osteoblast cells whereas Moresco cells had significantly lower expression. (D) Both Abrams and D17 cell lines showed high expression of PAX6 mRNA compare to canine osteoblast cells. All mRNA expressions studies were equilibrated with the HPRT housekeeping gene. Errors bars represent S.D., statistical analysis was performed using one-way ANOVA with post-hoc Tukey's test. Significance denoted as: * p<0.05, * * p<0.01, * * * p<0.001.
Figure 2.
Constitutive expression of GLI proteins in canine OSA cell lines.
Western Blot analyses demonstrating variable protein expression of GLI1 and GLI2 in canine OSA cell lines. Actin served as the internal loading control.
Figure 3.
Inhibitory effects of the GLI-inhibitor GANT61 on the colony formation and viability of canine osteosarcoma cell lines.
Canine OSA cell lines were treated with five different concentrations of the GLI-inhibitor GANT61. GANT61 decreased colony formation in (A) Abrams, (B) D17, and (C) Moresco cell lines. (D) GANT61 also reduced cell viability, as determined by MTS assay, in all cell lines. D17 cells appeared most sensitive to the effects of GANT61. Errors bars represent S.D.
Figure 4.
Expression of GLI1 target genes after GANT61 treatments in canine D17 cells.
Canine D17 OSA cells were treated with 12 µM of GANT61 for 96 hrs and relative expression was determined by Real Time RT-PCR. (A–C) GANT61 significantly decreased expression of GLI1, GLI2, and PTCH1 mRNA compared to untreated (UT) and vehicle DMSO treated cells. (D) Treatment of D17 cells with GANT61 significantly decreased expression of PAX6 mRNA. All mRNA expression studies were equilibrated with the housekeeping gene HPRT. Errors bars represent S.D., statistical analysis was performed using one-way ANOVA with post-hoc Tukey's test and significant values denoted as: * p<0.05, * * p<0.01, * * * p<0.001.
Figure 5.
Protein expression of Hh target genes after GANT61 treatments in canine D17 cells.
Canine D17 OSA cells were treated with 20 µM of GANT61 for 96 hrs and expression was determined by Western Blot analysis. GANT61 significantly decreased expression of GLI1, GLI2, PTCH1, and PAX6 compared to vehicle DMSO treated cells. Actin served as the internal loading control.
Figure 6.
Comparative transcript expression of GLI1 and GLI2 in canine Moresco OSA cells after GANT61 treatment.
(A–B) Canine Moresco OSA cells were treated with 12 µM of GANT61 for 96 hrs and expression levels of GLI1 and GLI2 were determined by Real Time RT-PCR. GANT61 significantly inhibited expression of GLI1 and GLI2 mRNA compared to untreated (UT) or vehicle DMSO treated cells. Errors bars represent S.D., statistical analysis was performed using one-way ANOVA and post-hoc Tukey's test and significance denoted as: * p<0.05, * * p<0.01, * * * p<0.001.