(A) Diagrammatic representation of the experimental procedure to analyze the effects of prolonged fasting (PF, 48 hr) during six cycles of cyclophosphamide chemotherapy (CP, 200 mg/kg, i.p.).

(B) Survival curve with vertical dashed lines indicating the prechemo fasting period; p < 0.01, log-rank (Mantel-Cox) test; n = 20 (ten male and ten female).

(C) DNA damage measurement (olive tail moment) in bone marrow (BM) cells (day 81, sixth recovery phase).

(D) Apoptosis measurement (TUNEL assay) in HSCs and MPP (day 81, sixth recovery phase).

∗p < 0.05, two-way ANOVA, comparing CP versus PF + CP during the recovery phase, n = 12 (six male and six female); L/M ratio of peripheral blood (PB) is defined as number of lymphocytes divided by number of myeloid cells (i.e., granulocytes and monocytes). See also (E) Hematological profile of mice. Total white blood cell (WBC), lymphocyte counts, and lymphoid/myeloid ratio (L/M) in mice treated with six cycles of CP (200 mg/kg, i.p.). Each point represents the mean ± SEM; horizontal dashed lines indicate the ranges of baseline values;p < 0.05, two-way ANOVA, comparing CP versus PF + CP during the recovery phase, n = 12 (six male and six female); L/M ratio of peripheral blood (PB) is defined as number of lymphocytes divided by number of myeloid cells (i.e., granulocytes and monocytes). See also Figures S1 F and S1G.

(F) Hematological profile of human subjects. Lymphocyte counts and lymphoid/myeloid ratio (L/M) in patients undergoing two cycles (C1 and C2) of platinum-based doublet chemotherapy in combination with either 24 or 72 hr (48 before and 24 hr after chemo) prolonged fasting; D1 and D8 indicate the first day (before chemo) and eighth day after each chemotherapy cycle; each point represents the mean ± SEM; ∗∗p < 0.01, two-way ANOVA; sample size is indicated in parentheses.

(G) FACS analysis of hematopoietic stem and progenitor cells (day 84, end of sixth cycle); horizontal dashed lines indicate the baseline value.

(H) Proportion of the lymphoid-biased (Ly-HSC), balanced (Bala-HSC), and of the myeloid-biased (My-HSC) hematopoietic stem cells. The markers used are lower side population of LSK (lower-SPLSK) for My-HSC, middle-SPLSK for Bala-HSC, and upper-SPLSK for Ly-HSC. The lower panels show a magnification of the SP population in the upper panels. ∗p < 0.05, one-way ANOVA comparing to AL.

(I) BM cells collected from mice treated with either CP or PF + CP were transplanted into the recipient mice. The chimerism of donor-derived cells in PB and that in BM was determined 16 weeks after primary BM transplantation. The ratio of lymphocytes to myeloid cells (L/M) in the reconstituted blood was also measured.

For (G) and (I), n = 6–10 per group, ∗p < 0.05, ∗∗p < 0.01, t test comparing the PF with the nonfasted control group both in combination with cyclophosphamide treatment. Error bars represent SEM.