Figure 2. Varied circadian periods and phases in individual cells of the Sik3^-/- suprachiasmatic nucleus (SCN).

Time-lapse single-cell bioluminescence images of ex vivo SCN explant cultures of (a) Sik3^+/+; Per2^Luc/Luc and (e) Sik3^-/-; Per2 ^Luc/Luc mice were used for the following analyses: Period distribution on SCN slices of Sik3^+/+ (b) and Sik3^-/- (f). Acrophase distribution on SCN slices of Sik3^+/+ (c) and Sik3^-/- (g). Time series of the bioluminescence signals observed on a Sik3^+/+ SCN slice along the bold line indicated in a) (d), and those from a Sik3^+/+ SCN slice along the bold line indicated in (b) (h). The original signals were smoothed via a moving average filter, and then normalized. (i) Period distribution in representative Sik3^+/+ and control slices. p<0.0001 vs. Sik3^-/- (test for equality of variance, F-test). (j) Acrophase distribution of representative Sik3^+/+ and Sik3^-/- slices. p<0.0001 vs. Sik3^-/- (F-test). (k) and l) Representative Bmal1-luc rhythms of Sik3-KD NIH3T3 (sh #2 and #3, red) and control cells (blue). (m) Averaged sequential half-periods (peak-to-trough or trough-to-peak) of Bmal1-luc rhythms in KD and control NIH3T3 cells as shown in (k and l) (n = 4 per group). Numbers (1–5) on the x-axis present the first half-period (hours from first trough to second peak) to the fifth half-period (hours from third trough to fourth peak).

Figure 2—figure supplement 1. Sik3 knockdown cells exhibit shortened and unstable period of circadian Per2 ^Lucc rhythms.

(a) A representative Bmal1-luc bioluminescence rhythms of NIH3T3 cells transfected with Sik3-KD (sh #1 or sh #4, red) and control shRNA (blue) (n = 4) as shown in Figure 2k and l. (b) Average circadian periods from first trough to third trough of individual experiments as shown in (b) and Figure 2k–l. (n = 4, ***p<0.001 vs. controls by Dunnett’s test) (c) Averaged sequential half-periods (peak-to-trough or trough-to-peak) of Bmal1-luc rhythms in KD and control NIH3T3 cells as shown in (k and l) (n = 4 per group). Numbers (1–5) on the x-axis present the first half-period (hours from first trough to second peak) to the fifth half-period (hours from third trough to fourth peak).

Figure 2—video 1. Time-lapse bioluminescence imaging of a coronal SCN slice of a Sik3^+/+; Per2 ^Luc/Luc mouse.

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Exposure time for a single image was 20 min, and time-lapse images were captured for 4 consecutive days. Per2 ^Luc rhythms are well synchronized in majority of the cells in the SCN.

Figure 2—video 2. Time-lapse bioluminescence imaging of a SCN slice of a Sik3^-/-; Per2 ^Luc/Luc mouse.

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Exposure time for a single image was 55 min, and time-lapse images were captured for 3 consecutive days. Note that Per2 ^Luc rhythms were desynchronized among individual cells in the SCN.