Figure 7: JIB-04 promotes GLI1 decay in human cancer cells.

(a) Immunoblot detecting endogenous GLI1 protein in lysates of human lung carcinoma cells (A549) and human rhabdomyosarcoma cells (RMS13). Cells were treated with 10 μM JIB-04 for 24 h. Shown is a representative result of two independent experiments. (b) Endogenous GLI1 protein stability assay in RMS13 cells exposed to JIB-04 (10 μM) and cycloheximide (CHX, 100 μg ml⁻¹) for the indicated time periods. Shown is a representative result of two independent experiments. Numbers indicate the GLI1 band intensity of the experiment shown normalized to actin. (c) Western blot detecting endogenous GLI1 protein in RMS13 cell lysates. Cells have been treated with the indicated compounds for 8 h. MG132: 10 μM; JIB-04: 10 μM. Shown is a representative result of three independent experiments. Numbers indicate the GLI1 band intensity of the experiment shown normalized to actin. (d) Western blot detecting endogenous GLI1 in Sufu−/− MEF cells and in Sufu−/− MEF cells stably expressing SUFU (Sufu−/−^[SUFU] cells). Cells were treated for 8 h with the indicated compounds. SAG: 100 nM; JIB-04: 1, 5, 10 μM. The BRD4 and GLI inhibitor JQ1 (ref. 45) was used as a positive control (1 μM). Shown is a representative result of three independent experiments. (e) GLI1 immunoblot of A549 xenograft samples receiving either solvent or JIB-04 (55 mg kg⁻¹ by oral gavage two to three times weekly for 1 month). (f) Quantification of GLI1 western blot bands (normalized to actin) shown in e. (g) Murine and human Hh pathway gene expression in A549 xenograft samples as measured by species-specific qPCR (mean of n=7±s.d., significance calculated using two-way analysis of variance). (h) Scheme depicting the major findings of this manuscript. DYRK1A can stimulate GLI1 activity by direct phosphorylation (left side). In contrast, DYRK1A is able to repress GLI1 activity through an indirect mechanism involving the actin cytoskeleton and its regulators. *P<0.05; **P<0.005; ***P<0.0005 (Student’s t-test).