Figure 6: Sp1-dependent activation of Cx43 expression by Rev-erbα.

(a) Sequences including Sp1 sites are indispensable for Cx43 promoter activation by Rev-erbα. *P<0.001 versus the control of each construct and †P<0.001 versus −54 (without Sp1 sequences) construct by two-way ANOVA with Bonferroni's post hoc test (n=3 for each). (b–e) Rev-erbα and Sp1 activate Cx43 expression using Sp1 sites. (b) Diagram of Cx43 promoter sequences including three Sp1 sites, labelled as Sp1A, B and C. The asterisk indicates corresponding nucleotide sequences among humans, rats and mice. (c) Dose-dependent activation of Cx43 transcription by Sp1 and Rev-erbα with Sp1. *P<0.001 versus the value -without Sp1 and Rev-erbα, and †P<0.001 by one-way ANOVA with Tukey's post hoc test (n=3 for each). (d) Immunoblot analysis of the effect of Sp1 and Rev-erbα on expression of Cx43 and Bmal1 (control of negative regulatory effect by Rev-erbα). (e) Impaired activation of pCx43 with the Sp1 sites mutation by Sp1 and Rev-erbα. *P<0.001 versus the controls of each construct, and †P<0.001 versus the MutC construct by two-way ANOVA with Bonferroni's post hoc test (n=3 for each group). Error bars represent s.d. in a, c and e. Cells used were HEK293T in all transfection experiments. The control without Rev-erbα and Sp1 was set as 1 in a, c and e. One representative of two experiments with similar results is shown in a, c, d and e.