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. 2021 Oct 18;10:e62469. doi: 10.7554/eLife.62469

Figure 3. Conditional myClock-KO mice show circadian pattern in mortality by endotoxic shock.

(A) and (B) Reduced levels of Clock mRNA and protein in myeloid lineage cells of myClock-KO mice. (A) Mean values of normalized mRNA expression values of time-series shown in (C) (n=21 mice per condition, p<0.0001, t-test). (B) Protein levels by immunoblot in peritoneal cavity cells and liver of myClock-KO and Clockflox/flox control mice (n=3). (C) Relative mRNA levels of selected clock genes in peritoneal macrophages from LysM-Cre (brown circles) or myClock-KO (red circles) mice, both kept in DD, at indicated circadian times. Phase and amplitude information are depicted in (D) as analyzed by Chronolyse. Non-significant circadian expression (p>0.05) are depicted in light red (myClock-KO) or light brown (LysM-Cre control). (E) Representative bioluminescence recordings of peritoneal macrophages, SCN or lung tissue from myClock-KO or wild-type mice crossed with PER2:Luc reporter mice (color coding as before). Black arrow indicates time of re-synchronization by dexamethasone treatment (detrended data). (F) Circadian pattern in endotoxic shock mortality despite deficiency of CLOCK in myeloid lineage cells. Mice (DD, n=10–14 per time point) were challenged with half-lethal doses of LPS (30 mg/kg, i.p.) at indicated time points. Mortality was assessed 60 hr after LPS injection. Statistic were performed as in Figure 1C (p=0.005, gray shaded area indicates 95% confidence interval). (G) Reduced mean mortality (at 30 mg/kg LPS) in mice deficient of myeloid CLOCK (n=103) compared to control strains LysM-Cre (n=39) or C57Bl/6 (wild-type, n=40). First two bars where re-plotted from Figure 2C. Error bars represent 95% confidence intervals (* p=0.0192, *** p = 0.0001).

Figure 3—source data 1. Raw imaging data files from western blots.

Figure 3.

Figure 3—figure supplement 1. Phenotypic and molecular characterization of myClock-KO mice.

Figure 3—figure supplement 1.

Phenotypic and molecular characterization of myClock-KO mice. (A) Locomotor activity recordings from control (LysM-Cre and Clock-flox) and myClock-KO mice. Representative examples are shown and quantified (n=7–13) in (B) for circadian periods as well as overall activity. (C) Prototypical genotyping results for Clockflox mice. (D) Relative mRNA levels of selected clock genes (complementing data of Figure 3C) in peritoneal macrophages from LysM-Cre (brown circles) or myClock-KO (red circles) mice at indicated circadian times. Phase and amplitude information are depicted in (E) as analyzed by Chronolyse. Non-significant circadian expressions (p>0.05) are indicated by light shaded circles for both myClock-KO and LysM-Cre control. (F) Kaplan-Meier survival curve of mice kept in DD and challenged with LPS (30 mg/kg, i.p.). Gray line refers to C57Bl/6 mice (wild-type control), brown line to LysM-Cre control and red line to myClock-KO mice. Data have been aligned to time after challenge with LPS, irrespective of time of day of challenge (data are from experiments shown in Figure 1C and Figure 3F,G, respectively). Survival curves of wild-type and myClock-KO differ significantly (p<0.0001, log-rank test).