Fig 2: Effect of CXCR4 and CXCR7 knockdown on spontaneous beating of hiPSC-CMs. Spontaneously beating hiPSC-CMs (embryoid bodies) were counted starting at 5 days after differentiation. Data is shown as mean ± SEM and statistical analysis was done by two-way ANOVA followed by Tukey’s post-test (n = 3; *P < 0.05, **P < 0.01, #P < 0.001 vs. control).

Fig 3: Relevance of Rhythmic Leukocyte Trafficking in Inflammation, Leukemia, and Humans(A) Blood leukocyte numbers after acute treatment without (ctrl) or with LPS in combination with functional blocking antibodies directed against the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA followed by Dunnett comparison to the LPS group and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(B) Overview of functional blocking effects on leukocyte subsets in blood after LPS treatment targeting the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA).(C) Numbers of circulating blasts present in the blood of C57BL/6J CD45.1 recipients at midday 1 week after engraftment at ZT1 and ZT13 with mouse C1498 (AML) or BS50 (B-ALL) cells (n = 7 or 8 mice; Mann-Whitney test).(D) Numbers of circulating blasts present in the blood of NSG recipient mice at midday 1 week after engraftment at ZT1 and ZT13 with human NALM-6 B-ALL cells (n = 8 mice; unpaired Student’s t test).(E) Oscillation of blood B cell numbers in human blood (n = 8 subjects; repeated-measures one-way ANOVA).(F) CXCR4 expression on human B cells over 24 hr (n = 8 subjects; repeated-measures one-way ANOVA).(G) Transendothelial migration (TEM) capacity of human primary B cells harvested from three donors at 11 a.m. and 7 p.m. across HUVECs. Numbers are normalized to 11 a.m. levels (n = 4 assays; unpaired Student’s t test).(H and I) Blocking efficacy of AMD3100 (H) or an anti-LFA-1 antibody (I) on TEM capacity of human primary B cells harvested at 11 a.m. and 7 p.m. Numbers are normalized to and compared with those of vehicle and isotype controls, respectively (n = 3 donors; unpaired Student’s t test).(J and K) Example of the TEM capacity of human B cells from one patient at 11 a.m. and 7 p.m. after AMD3100 (J) or anti-LFA-1 treatment (K) plotted over time (n = 4 assays; two-way ANOVA with Tukey post-test).*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; #, ##, ###, #### indicate significance levels analogous to those of the LPS groups. All data are represented as mean ± SEM. ns, not significant. See also Figure S7.

Fig 4: Chronopharmacological Targeting of Leukocyte Homing to Tissues(A–H) Adoptive transfer of donor cells to the bone marrow (A and B), lymph node (C and D), spleen (E and F), liver (G), and lung (H) of recipients treated with functional blocking antibodies or antagonists directed against the indicated molecules at ZT13. Cell numbers are normalized to control numbers (n = 4–8 mice; one-way ANOVA followed by Dunnett comparison to the control group). Localization of transferred cells in the bone marrow (B), lymph node (D), and spleen (F) is normalized to control localization (n = 4–8 mice; one-way ANOVA followed by Dunnett comparison to the control group).(I) Images and quantification of localization of donor cells in bone marrow after CXCR4 blockade. Boxes indicate exemplary cells whose localization within tissues is additionally shown in the z direction (n = 3 mice; unpaired Student’s t test). Scale bars: 10 µm.(J) Overview of functional blocking effects on leukocyte recruitment to organs, targeting the indicated molecules (n = 4–8 mice; one-way ANOVA followed by Dunnett comparison to the control group).*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data are represented as mean ± SEM. See also Figure S5.

Fig 5: Leukocyte-Subset-Specific Oscillations in Pro-migratory Molecules(A) Map of rhythmic protein expression of adhesion molecules and chemokine receptors in blood leukocyte subsets (n = 3–6 mice with 4–6 time points measured each; one-way ANOVA).(B) Adoptive transfer of ZT1 and ZT13 donor cells to recipients treated with functional blocking antibodies directed against the indicated molecules at ZT1 and ZT13. Cell numbers are normalized to ZT1 and ZT13 controls (n = 3–12 mice; one-way ANOVA followed by Dunnett comparison to control groups and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(C) Adoptive transfer of donor cells to recipients treated with antagonists against the indicated molecules at ZT1 and ZT13 (n = 3–10 mice; one-way ANOVA followed by Dunnett comparison to control groups and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(D) Fold change of donor cells remaining in recipient blood at ZT1 and ZT13 after anti-VCAM-1 and anti-ICAM-1 antibody treatment, respectively, in comparison with numbers of isotype antibody controls. (n = 3 or 4 mice; one-way ANOVA followed by Dunnett comparison to control groups and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(E) Endogenous blood leukocyte numbers after CXCR4 antagonist treatment (n = 3 or 4 mice; one-way ANOVA followed by Dunnett comparison to control groups and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(F) Overview of functional blocking effects on adoptively transferred leukocyte subsets in blood targeting the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA followed by Dunnett comparison to control groups).*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; #, ##, ###, #### indicate significance levels analogous to those of control groups. All data are represented as mean ± SEM. ns, not significant. See also Figure S3 and Table S2.