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. 2014 Jul 16;28(9):1471–1486. doi: 10.1210/me.2014-1060

Figure 6.

Figure 6.

Ccdc80 is a novel βcat-regulated gene in adrenocortical cells. A, Section in situ hybridization for Ccdc80 expression in the adult mouse adrenal. B, ATCL7 cells were treated with 0.5μM BIO or vehicle for 18 hours in low-serum media and harvested. Changes in Ccdc80 expression were assessed by qRT-PCR. Values represent the mean ± SD, where vehicle-treated cells were normalized to 1 (n = 4). **, P < .005. C, ATCL7 cells were electroporated with empty vector, βcatS33Y, or dnTCF4E alone or in combination. At 48 hours after transfection, cells were harvested and changes in Axin2 expression (left) and Ccdc80 expression (right) were measured by qRT-PCR. Values represent the mean ± SD (n = 4), where vehicle-treated cells were normalized to 1. D, Diagram of Ccdc80-luciferase reporter construct; −1957 to +814 upstream of the transcription start site of Ccdc80 was cloned into pGL3b luciferase reporter plasmid. Four putative Tcf/Lef binding sites identified using Genomatix MatInspector are labeled #1 to #4. E, 293T cells were transfected with wild-type Ccdc80-luciferase construct or constructs with each individual Tcf binding site mutated (#1–4) and Renilla luciferase internal control construct for 24 hours. Cells were treated with 2μM BIO or vehicle for 24 hours before passive lysis. Luciferase activity was measured and normalized to Renilla luciferase for transfection efficiency. Values represent the mean ± SD (n = 4), where wild-type vehicle-treated cells were normalized to 1. F, ATCL7 cells were electroporated with wild-type Ccdc80-luciferase construct or constructs with each individual Tcf binding site mutated (#1–4) and Renilla luciferase internal control construct for 24 hours. Cells were treated with 0.5μM BIO or vehicle for 24 hours before passive lysis. Luciferase activity was measured and normalized to Renilla luciferase for transfection efficiency. Values represent the mean ± SD, where wild-type vehicle-treated cells were normalized to 1 (n = 4). ***, P < .0005; **, P < .005; *, P < .05; n.s., not significant.