Figure 4: Oscillation of the circadian clock, Cx43 and gap junction function in bladder muscle cells without systemic control.

(a) Oscillation of luminescence in bladder ex vivo slice culture obtained from mPer2^Luciferase knock-in (Per2::luc) mice. The period of oscillation was 24.92±0.56 (mean±s.d.) (n=10). The muscle layer of the bladder is shown by alpha smooth muscle actin (αSMA) immunostaining. m, muscle. The scale bar indicates 100 μm. The oscillation of luminescence is also shown by a movie in Supplementary Movie 1. (b) Temporal variation of Per2, Bmal1 and Cx43 mRNA levels in serum-shocked rat BSMC. *P<0.01 against the nadir of each genes' mRNA levels (time 12 for Per2, time 48 for Bmal1 and time 64 for Cx43) by one-way ANOVA with Dunnett's post hoc test (n=3–6). SS, serum shock. For relative levels, the values before serum shock were set as 1. (c) Immunoblots showing temporal changes in Cx43 protein levels with αSMA as a loading control in serum-shocked rat BSMC. (d) Immunostaining of Cx43 at times 12, 24, 36 and 48 h in serum-shocked rat BSMC (red, Cx43; blue, DAPI). Arrow heads indicate typical plaques of gap junctions. Representative images of two replicate experiments with similar results are shown in c and d. (e) Oscillation of gap junction function evaluated by Lucifer yellow microinjection in serum-shocked rat BSMC. One representative photograph of each time point (green, Lucifer yellow; blue, Hoechst 33342) and overall quantification of the degree of dye-coupling (n=6–9, a total of 71 injections) is shown. *P<0.05 and **P<0.01 versus time 24 h by one-way ANOVA with Tukey-Kramer's post hoc test. Similar significant differences were obtained in two independent experiments. Error bars represent s.e.m. in b and e. Scale bars in d and e indicate 100 μm.