Supplementary Experimental Procedures:
Rota-rod. Mice were tested on a five station Rota-Rod treadmill for mice (MED Associates, St. Albans, VT). The rotation speed started at 4 rpm and increased by 10 rpm per min to a maximum speed of 40 rpm. Latency to fall from the rod wasrecorded. If an animal fell in <10 sec, it was placed back on the rod for a maximum of five times. Each mouse was subjected to four trials per day for 3 consecutive days, with at least 1 hr recovery time between trials. The data presented was the average of 4 trials of each day.
Hanging Wire.Mice were tested for neuromuscular abnormalities (balance and grip strength) by suspending them from wire bars (10 × 20 cm area, 1 mm in diameter spaced 1 cm apart). Latency to fall (maximum, 3 min) was measured after a mouse was placed on the bars and turned upside down (height, 30 cm). Mice that fell in <10 second were given a second trial. The test was repeated twice with interval of 48 hr. The averages of the two tests were plotted.
Open-Field Activity.Mice were placed in a novel open-field box (MED Associates, St. Albans, VT; 42.5 × 42.5 cm) and tracked by an array of infrared beams for the duration of 60 min. The infrared beams breaks were accumulated every 10 min.
Legends for the supplementary figures:
Figure S1. The wheel running (A) and spontaneous activity patterns of WT and PK2-/- mice under a LD cycle.
For each animal, wheel running counts or infrared beam breaks were accumulated in 1-hr bins for 5 days under LD and then averaged to produce one 24-hr profile. The data from a group of WT (blue closed circles) orPK2-/- (red open circles) mice were then averaged and plotted with respect to ZT. ibb, infrared beam breaks.
Figure S2.Normal motor functionsof thePK2-/- mice.
PK2-/-and WT mice showed indistinguishable behaviors in the Rota-Rod (A), Hanging Wire (B) and open field (C) tests. Blue, WT; Red, PK2-/-.Each data represents mean ± SEM of 8-12 mice.
Figure S3. The amplitude of core body temperature under LD was significantly reduced in thePK2-/-mice.
A.The amplitude of core body temperature was the difference between peak values and the means of light phase. *, P < 0.02, Student’s t test, n = 5 mice/group. Tb, body temperature.B-C, representative core body temperature recordings of a wild-type mouse (B) and a PK2-/- mouse (C) under 7-day DD condition. Their Fourier analysis were shown at the right panels. The asterisks indicated the relative FFT power of 24 hr/cycle.
Figure S4.The temporal oscillation of total sleep patterns was significantly reduced in thePK2-/-mice.
Total sleep (TS) values as percentage of recording time werecalculated in 2-hr bins. The data of 2 LD cycles (A) and 2 DD cycles (B) from wild-type (11 mice, closed circles) or PK2-/- (11 mice, open circles) mice were then averaged and plotted as mean ± SEM. Thick continuous lines (wild-type, blue lines; PK2-/-, red lines) indicate the best-fit cosine waves used to calculate the amplitude of the rhythms. Clear horizontal bars indicate the light periods, black bars indicate the dark periods, and shaded bars indicate the subjective light periods under DD. Please note that the major reduction of TS in the PK2-/- miceoccurred in the light or subject light periods. (C)The amplitude (equal to the peak value minus trough value) calculated from simulated cosine waves under LD (A) and DD (B) were significantly different between wild-type and PK2-/- mice(* P < 0.001, Student’st test). The cosine simulation and calculations were performed by Prism 4.4 software.