Relationship between GLI1 expression and GLI1 downstream targets. (A) GLI1, PTCH1, E2F1, AKT1, and BIM transcript levels in CLL cells expressing or lacking GLI1 (GLI1⁺, n = 28; GLI1^–, n = 21). The Mann-Whitney U test was used to calculate the P value indicated at the top. (B) Representative immunoblot analyses of CLL cells with or without GLI1 expression (GLI1⁺, n = 28; GLI1^–, n = 21), as indicated at the top. Each lane represents a separate case. The membranes were probed with a monoclonal antibody specific for GLI1, PTCH1, E2F1, AKT1, pBIM, and BIM or β-actin as indicated on the left margin. The expression of β-actin was used to normalized GLI1, PTCH1, E2F1, AKT1, and BIM expression levels. The ratios of the band densities for each case are provided at the bottom of each blot and presented in the dot-plots in panel C. The ratios of the band densities for each case of pBIM_EL^S87/BIM_EL is indicated at the bottom of the pBIM_EL^S87 blot and represented in the dot-plots in panel C. The same GLI1⁺ CLL protein lysate sample was used as positive control in all gels. (C) Densitometry analysis quantifying the protein expression levels of GLI1 and its downstream targets in all 49 cases with CLL cells with or without GLI1 expression. The horizontal bar provides the mean ratio observed in each group. The Mann-Whitney U test was used to calculate the P value indicated at the top. (D) Schematic representation of the consequences of GLI1 upregulation on its downstream targets. (E) Relative viability of CLL cells treated with control siRNA (siCTR) or GLI1-specific siRNA (siGLI1) as indicated. (F) Immunoblot analyses for GLI1 using lysates of CLL cells expressing GLI1 treated with siCTR or siGLI1. Data from 2 representative patients are presented. (G) Relative viability of CLL cells expressing GLI1 with (GLI1⁺ Hh_MU, n = 6; red squares) or without (GLI1⁺ Hh_WT N = 3, gray squares) Hh mutations or lacking GLI1 expression with (GLI1^– Hh_MU, n = 3; blue circles) or without (GLI1^– Hh_WT, n = 6; yellow circles) Hh mutations treated for 24 hours with 5 or 10 μM of GANT61. Data are shown as mean ± standard deviation. (H) Immunoblot analyses of CLL cells expressing GLI1 and treated for 24 hours with 10 μM of GANT61. The membranes were probed with a monoclonal antibody specific for GLI1, pBIM, and BIM or β-actin as indicated on the left margin. The expression of β-actin was used to normalized GLI1 and BIM expression level. The ratios of the band densities for each case are provided at the bottom of each blot. The ratios of the band densities for each case of pBIM_EL^S87/BIM_EL are indicated at the bottom of the pBIM_EL^S87 blot. Data from 2 representative patients are presented.