Figure 2
Figure 2. GLI1 transcriptional activity is modulated by IKKβ. (A) Immunoblot analysis of GLI1 in indicated cells stimulated with or without TNFα (20 μg/mL) for indicated time periods. Phosphorylation of the P65 (Ser-536) subunit of NF-κB confirmed IKKβ-mediated activation of the NF-κB pathway in the TNFα-stimulated cells. (B) 8X GLI1 luciferase and Renilla constructs were cotransfected in indicated cells for 48 hours and subjected to luciferase reporter assay. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (*P < .05). (C) Immunoblot analysis of GLI1 in 293T cells transiently transfected with constructs carrying FL-GLI1 and the IKKβ or IKKβ-KD. (D) GLI1 luciferase reporter analysis of 293T cells transiently transfected with constructs carrying vector, IKKβ, or FL-GLI1 and IKKβ. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (**P < .005). (E) Immunoblot analysis of GLI1 in DLBCL cells treated with or without TPCA1 for indicated time periods. (F) GLI1 luciferase reporter analysis of HBL1 cells treated with or without TPCA1. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (** P < .005). (G) HBL1 cells were transduced with lentiviral particles expressing shRNA-targeting luciferase (control) and IKKβ shRNAs. The transduced cells were selected with puromycin and subjected to immunoblotting. The same cells as described in (G) were used for qRT-PCR analysis of BCL2, GLI1, and PTCH1 mRNA expression. Results are normalized to GAPDH mRNA level and expressed as fold changes in mRNA expression compared with control. Data represent the mean and standard deviation of 2 independent experiments (*P < .05; ***P < .0005).

GLI1 transcriptional activity is modulated by IKKβ. (A) Immunoblot analysis of GLI1 in indicated cells stimulated with or without TNFα (20 μg/mL) for indicated time periods. Phosphorylation of the P65 (Ser-536) subunit of NF-κB confirmed IKKβ-mediated activation of the NF-κB pathway in the TNFα-stimulated cells. (B) 8X GLI1 luciferase and Renilla constructs were cotransfected in indicated cells for 48 hours and subjected to luciferase reporter assay. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (*P < .05). (C) Immunoblot analysis of GLI1 in 293T cells transiently transfected with constructs carrying FL-GLI1 and the IKKβ or IKKβ-KD. (D) GLI1 luciferase reporter analysis of 293T cells transiently transfected with constructs carrying vector, IKKβ, or FL-GLI1 and IKKβ. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (**P < .005). (E) Immunoblot analysis of GLI1 in DLBCL cells treated with or without TPCA1 for indicated time periods. (F) GLI1 luciferase reporter analysis of HBL1 cells treated with or without TPCA1. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 3 independent experiments (** P < .005). (G) HBL1 cells were transduced with lentiviral particles expressing shRNA-targeting luciferase (control) and IKKβ shRNAs. The transduced cells were selected with puromycin and subjected to immunoblotting. The same cells as described in (G) were used for qRT-PCR analysis of BCL2, GLI1, and PTCH1 mRNA expression. Results are normalized to GAPDH mRNA level and expressed as fold changes in mRNA expression compared with control. Data represent the mean and standard deviation of 2 independent experiments (*P < .05; ***P < .0005).

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