Figure 2: Fasting returns Ly-6C^hi monocytes back from circulation to the bone marrow.

A. Ly-6C^hi monocytes isolated from GFP positive mice were injected intravenously into wild type mice directly prior to initiation of a 24h fast or feeding ad libitum.

B. Representative flow cytometry plots of GFP positive monocytes after feeding ad libitum and fasting in the blood. Adoptively transferred monocytes are defined as CD45⁺ GFP⁺. (n=5–6 per group, two independent experiments combined)

C. Representative flow cytometry plots of GFP positive monocytes after feeding ad libitum and fasting in the bone marrow (BM). Adoptively transferred monocytes are defined as CD45⁺ GFP⁺. (n=5–6 per group, two independent experiments combined)

D. Relative decrease of GFP positive monocytes in the blood and relative increase of GFP positive monocytes in the bone marrow after 24h fasting compared to fed control (n=5–6 per group, two independent experiments combined). Unpaired t test.

E. Relative expression of target genes of sorted blood Ly-6C^hi monocytes after 24h fasting normalized to fed control (n=3 mice per group). Unpaired t test.

F. Circulating Ly-6C^hi monocytes measured at indicated timepoints in Cxcr4^fl/fl Lyz2^Cre and Cxcr4^fl/fl under fed and fasted conditions (n=4–10, timepoints are independent experiments). Start of the fast is indicated by orange zeitgeber (ZT). Two-way ANOVA.

G. Plasma corticosterone (CORT) in Cxcr4^fl/fl under fed and fasted conditions at indicated timepoints (n=4–8, timepoints are independent experiments). Start of the fast is indicated by orange zeitgeber (ZT). Two-way ANOVA.

H. Relative expression of Cxcr4 on sorted blood Ly-6C^hi monocytes after incubation with CORT (n=10 mice pooled for sort, n=3 technical replicates per group). Unpaired t test.

I. Mice underwent bilateral adrenalectomy (ADX) or sham surgery and were allowed to recover for 2 weeks, prior to submission to feeding ad libitum or a 12h fast. Blood Ly-6C^hi monocytes in mice 2 weeks post ADX or sham surgery after fasting compared to fed ad libitum (n=4–6 per group). Unpaired t test.

J. Blood Ly-6C^hi monocytes measured at indicated times in Nr3c1^fl/fl Lyz2^Cre and Nr3c1^fl/fl under fed and fasted conditions (n=4–11, timepoints areindependent experiments). Start of the fast is indicated by orange ZT. Two-way ANOVA.

K. Blood Ly-6C^hi monocytes in Crh^fl/fl Sim1^Cre and Crh^fl/fl after 4h of fasting compared to fed ad libitum (n=3–6 per group). Unpaired t test. Data presented as mean ±SEM, *p < 0.05, **p<0.01, ***p < 0.001. FI: Fluorescence intensity, BM: Bone marrow, CORT:

Corticosterone, ADX: Adrenalectomy. Please also see Figure S1.