Figure 1
H2O2 treatment-induced dose-dependent phase-changes in the PER2::LUC rhythm in MEF cells. (A, C) Representative detrended data of PER2::LUC bioluminescence from MEF cells treated with water as a vehicle or 0.01-2.0 mM H2O2. Treatment was performed at Circadian Time (CT; CT0 indicates the trough of bioluminescence and CT12 indicates the peak) 18 (A) and CT4 (C) after the first peak of the PER2::LUC rhythm (indicated by the arrow). (B, D) Phase changes of the third peak in each treatment group, compared with the peak of intact. Phase delay (B) was seen in the experiment (A), and phase advance (D) was seen in the experiment (C). (E) Amplitude of the third peak was compared by bioluminescence (photon/sec). (F) Number of cells were counted after 4 days of luminescent monitoring. All values are mean ± SEM (n = 7–8 per group for A, n = 4 per group for C). ***p < 0.001 (vs. vehicle) by Dunnett test, #p < 0.05 (vs. vehicle) by Dunn test. int (intact); veh (vehicle).
Figure 2
Phase response curve (PRC) of H2O2-induced PER2::LUC phase change in MEF cells. (A) Representative detrended data of PER2::LUC bioluminescence from MEF cells treated with 0.2 mM H2O2 at Circadian Time (CT; CT0 indicates the trough of bioluminescence and CT12 indicates the peal) 16, 20, and 22 (left panel), and CT4 and 8 (right panel). Arrows indicate the stimulation time points in each group. (B, C) PRC of PER2::LUC phase shift by 0.2 mM H2O2 (left panel) or vehicle (water, right panel). In the PRC, the peak change values of the third peak of bioluminescence of the intact and treated groups are indicated. All values are mean ± SEM (n = 4 for CT16, 20, 22, 28, and 32 in H2O2, n = 8 for CT12 and 24 in H2O2, and n = 4 for vehicle). *p < 0.05, **p < 0.01, ***p < 0.001 (vs. intact) by Sidak test.
Figure 3
Phase changes in the PER2::LUC rhythm by H2O2 treatment in vivo. (A) Experimental schedule. PER2::LUC mice were intraperitoneally (i.p.) injected with the vehicle (PBS) or H2O2 (0.1–0.5 M) (10 ml/kg of mouse body weight) at ZT4 for 3 consecutive days. Then, the PER2::LUC rhythm of peripheral tissues was detected by an in vivo imaging system. (B) Representative images of in vivo PER2::LUC bioluminescence in the kidney (upper panels) and in the liver and submandibular gland (sub gla) (lower panels). (C) Normalized PER2::LUC oscillation in each tissue in vehicle and H2O2 (0.5 M) groups. P value on the lower right side of each graph indicates the results of two-way ANOVA between the vehicle and H2O2 groups. *p < 0.05, **p < 0.01, ***p < 0.001 (vs. intact) by Sidak test. (D) Peak phases of peripheral PER2::LUC oscillation in each group. All values are mean ± SEM (n = 6 in the intact group, n = 5 in the PBS and 0.25% 0.5 M H2O2 groups, n = 3 in 0.1 M H2O2). *p < 0.05, **p < 0.01 (vs. vehicle) by Dunnett test. (E) Representative locomotor activity data in vehicle or H2O2 (0.5 M) treatment. Injection was performed at ZT4 for consecutive 3 days (indicated by arrow heads).
Figure 4
Phase changes in the PER2::LUC rhythm by hemin treatment in vivo. Experimental schedule was the same as Fig 3. Hemin (30 or 50 mg/kg, PBS for vehicle) was treated at ZT4 for 3 consecutive days, and then peripheral PER2::LUC rhythm was measured. (A) Normalized PER2::LUC oscillation in each tissue in vehicle and hemin (50 mg/kg) groups. P value on the lower right side of graph indicates the results of two-way ANOVA between the vehicle and hemin groups. (B) Peak phases of peripheral PER2::LUC oscillation in each group. All values are mean ± SEM (n = 5 in the PBS group and n = 6 in both hemin groups). ***p < 0.001 (vs. vehicle) by Dunnett test, #p < 0.05 (vs. vehicle) by Sidak test.