Figure 5: Rev-erbα upregulates Cx43 expression.

(a) Dose-dependent activation of Cx43 transcription by Rev-erbα in HEK293T cells (n=3 for each dose). (b) Impaired activation of Cx43 transcription by a mutant of Rev-erbα without 127–206 amino acids from the N-terminal (Rev-Mut) in HEK293T cells (n=3 for each dose). (c) Activation of Cx43 transcription by Rev-erbα in rat BSMC (n=6 for each dose). *P<0.01 versus Rev-erbα (−) by one-way ANOVA with Dunnett's post hoc test in a–c. Similar data obtained in three independent experiments for a and b, and in two independent experiments for c. (d) Suppression of Cx43 expression by knock-down of endogenous Rev-erbα in BSMC. Three types of Rev-erbα siRNAs, containing high (si-1), middle (si-2) and low (si-3) GC ratios or their controls containing corresponding GC ratios were transfected. (n=4). mRNA and protein expression (data of si-3) was normalized by 18 s ribosomal RNA and GAPDH, respectively. Interference of Rev-erbα mRNA significantly decreased mRNA expression of Cx43 and increased Bmal1 compared with their corresponding controls (F(1[DF for the treatment factor],18[error DF])=324 for Rev-erbα, 9.7 for Cx43 and 11.7 for Bmal1. *P<0.01 by two-way ANOVA). Temporal bladder Rev-erbα mRNA accumulation in WT and Cry-null mice (n=3). (e) and in rats under LD (n=5) and DD (n=2) conditions (f). *P<0.05 versus CT8 and **P<0.01 versus CT0, 16 and 20 in WT by one-way ANOVA with Tukey's post hoc test. No significant difference in Cry-null mice. MaxCorrs were WT, 0.98; Cry-null, 0.31; rats in LD, 0.84; in DD, 0.93. The maximal value of WT was set as 1. Error bars represent s.d. in a–f. For relative levels, Rev-erbα (−) was set as 1 in a–c.