Figure 7.

Pharmacological inhibition of calcineurin dampens circadian expression of PER and TIM proteins in wild-type fly heads and enhances their proteasomal degradation in cell culture. (A) Wild-type flies were fed on behavior food (5% sucrose and 2% agar) containing either 1 mM DMSO (vehicle control) or 1 μM CsA throughout the behavioral test, and their averaged actograms were double-plotted (n = 45–57). Locomotor activity peaks anticipatory to lights-on were indicated by red arrows. (B) Wild-type flies were entrained by three LD cycles on the behavior food containing either 1 mM DMSO or 1 μM CsA, transferred to DD, and then harvested at the indicated time points during the first DD cycle. Relative expression levels (%) of PER and TIM proteins were quantified in head extracts similarly as in Figure 2. Data represent average ± SEM (n = 2). * P < 0.05 vs. DMSO controls at the same time point as determined by Student’s t-test. (C and D) Drosophila S2 cells were transfected with the expression vector for V5-tagged PER or TIM proteins. Where indicated, 20 μM CsA, 20 μM tacrolimus (FK506), or 50 μM MG132 were incubated with transfected cells for 2 hr before harvest. Relative expression levels (%) of V5-tagged PER or TIM proteins were quantified in transfected cell extracts similarly as in Figure 2. Data represent average ± SEM (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 as determined by one-way ANOVA with Tukey’s post hoc test. CsA, cyclosporin A; CT, circadian time; DD, constant dark; LD, 12 hr light/12 hr dark.