Figure 2.

Manipulation of the PPP Affects Circadian Oscillations through NADPH

(A) Bioluminescence traces for Bmal1:luc U2OS cells treated with 50 μM dehydroepiandrosterone (DHEA) versus control (DMSO), followed by wash off after 96 hr. (mean values shown, n = 3–6)

(B) Quantifications of the period length from (A) before and after wash off (mean ± SEM, n = 3–6; two-tailed Student’s t test; ^∗∗∗p < 0.001).

(C) NADP⁺:NADPH ratio of cells treated with DHEA (mean ± SEM, n = 3–4; two-tailed Student’s t test; ^∗∗p < 0.01).

(D) Treatment of Bmal1:luc U2OS cells with 5 mM 6AN decreases the levels of total NAD, while incubation with 50 μM DHEA has no effect. NAD levels in presence of 6AN can be restored by addition of 500 μM NMN (control [DMSO] versus treated cells; mean ± SEM, n = 3–4; two-tailed Student’s t test; ^∗∗∗p < 0.001, ^∗∗p < 0.01).

(E) Treatment with 500 μM NMN does not restore NADP⁺:NADPH ratio to normal levels (two-tailed Student’s t test, control [DMSO] versus treated cells; mean ± SEM, n = 3–4, ^∗∗∗p < 0.001, ^∗∗p < 0.01).

(F) Bioluminescence traces for Bmal1:luc U2OS cells treated with 6AN, or 6AN and NMN, versus control (DMSO) (mean values shown, n = 8).

(G) Quantifications of the period length from (F) (two-tailed Student’s t test; mean ± SEM, n = 8, ^∗∗∗p < 0.001, ^∗p < 0.05).