Figure 3.
Figure 3. Relationship between GLI1 expression and Hh-pathway mutations. (A) GLI1 protein expression in CLL cells with missense mutations (n = 39) deduced as being benign or damaging according to PolyPhen-2. The horizontal bar in each group provides the mean level of GLI1 protein expression observed for each group. The Mann-Whitney U test was used to calculate the P value indicated at the top. (B) Immunoblot analyses of CLL cells and the U937 cell line transfected with empty vector (C–), wild-type SMO (SMOWT), or mutant SMO carrying P26S, which is a non-inactivating missense mutation (SMOMU), as indicated at the top. The membranes were probed with a monoclonal antibody mAb specific for GLI1, SMO, or β-actin as indicated on the left margin. The expression of β-actin was used to normalize GLI1 and SMO expression. The ratios of the band densities are provided at the bottom of each blot. (C) Immunoblot analyses for proteins indicated on the right using lysates of CLL cells that were treated with control siRNA (siCTR) or tumor suppressor–specific (siFBXW7 or siCREBBP or siBCOR) siRNA as indicated. (D) Densitometry analysis of immunoblot in panel E quantifying GLI1 protein expression levels for CLL sample with leukemia cells with or without Hh-pathway mutations (HhMU, n = 49; HhWT, n = 161). The horizontal bar in each group provides the mean level of GLI1 protein expression observed for each group. The Mann-Whitney U test was used to calculate the P value indicated at the top. (E) Immunoblot analyses of CLL cells with or without Hh-pathway mutations (HhMU), as indicated at the top. Each lane represents a separate case. The membranes were probed with a monoclonal antibody specific for GLI1 or β-actin, as indicated on the left margin. The density of the β-actin band was used to normalize band density for GLI1 in each sample. The ratios of the band densities of GLI1/β-actin for each sample are indicated at the bottom and presented in the dot-plots in panel D. The same protein lysate from JeKo-1 cells was used as a positive control (C+) in all gels. GLI1+ CLL samples were defined as those with ratios of the band densities of GLI1/β-actin >0.1.

Relationship between GLI1 expression and Hh-pathway mutations. (A) GLI1 protein expression in CLL cells with missense mutations (n = 39) deduced as being benign or damaging according to PolyPhen-2. The horizontal bar in each group provides the mean level of GLI1 protein expression observed for each group. The Mann-Whitney U test was used to calculate the P value indicated at the top. (B) Immunoblot analyses of CLL cells and the U937 cell line transfected with empty vector (C–), wild-type SMO (SMOWT), or mutant SMO carrying P26S, which is a non-inactivating missense mutation (SMOMU), as indicated at the top. The membranes were probed with a monoclonal antibody mAb specific for GLI1, SMO, or β-actin as indicated on the left margin. The expression of β-actin was used to normalize GLI1 and SMO expression. The ratios of the band densities are provided at the bottom of each blot. (C) Immunoblot analyses for proteins indicated on the right using lysates of CLL cells that were treated with control siRNA (siCTR) or tumor suppressor–specific (siFBXW7 or siCREBBP or siBCOR) siRNA as indicated. (D) Densitometry analysis of immunoblot in panel E quantifying GLI1 protein expression levels for CLL sample with leukemia cells with or without Hh-pathway mutations (HhMU, n = 49; HhWT, n = 161). The horizontal bar in each group provides the mean level of GLI1 protein expression observed for each group. The Mann-Whitney U test was used to calculate the P value indicated at the top. (E) Immunoblot analyses of CLL cells with or without Hh-pathway mutations (HhMU), as indicated at the top. Each lane represents a separate case. The membranes were probed with a monoclonal antibody specific for GLI1 or β-actin, as indicated on the left margin. The density of the β-actin band was used to normalize band density for GLI1 in each sample. The ratios of the band densities of GLI1/β-actin for each sample are indicated at the bottom and presented in the dot-plots in panel D. The same protein lysate from JeKo-1 cells was used as a positive control (C+) in all gels. GLI1+ CLL samples were defined as those with ratios of the band densities of GLI1/β-actin >0.1.

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