Extended Data Fig. 2: Daytime NADH elevation regulates genome-wide transcription of fatty acid metabolism genes through BMAL1 during TRF-CR.

a, (left) RNA-seq reads per 10 million sequenced reads (RPM) that align to LbNOX in LbNOX- vs null-transduced TRF-Reg (n = 6 per genotype) and TRF-CR (n = 6 for null; n = 5 for LbNOX) liver. (right) Representative in situ immunohistochemistry against FLAG-LbNOX in liver of null- and LbNOX-transduced mice. Experiments were performed in 4–6 mo old male C57BL6/J mice unless otherwise noted. b, Heatmap depicting log₂-FC in gene expression from TRF-CR and LbNOX in TRF-CR for genes within gluconeogenic and glycolytic gene ontology groups. c, Unbiased principal components analysis during the day (ZT4) or night (ZT16) of null- and LbNOX-expressing TRF-Reg or TRF-CR mice for genes that are differentially-expressed by TRF-CR in null mice at respective time points (DESeq2 p-adj<0.05) (n as in panel a). d, Log₂-transformed FC in expression of select fatty acid metabolism genes during TRF-CR (blue) (n = 6), following LbNOX-overexpression during TRF-CR (white) (n = 5), and in Bmal1 knockout liver (purple) (n = 3). e, 24-hour fasted body temperatures in 4–6 mo old female liver-specific Bmal1^fx/fx mice before and after retro-orbital administration of AAV8–TBG-iCre (n = 6) (left: ANOVA, p < 0.01; right: *p < 0.05 in paired, two-way student’s t test). f, Log₂-transformed FC in expression of genes within the methionine pathway for conditions (n as in panel d). g, (top) Summary of methionine pathway and (bottom) metabolomics of effect of TRF-CR and LbNOX in TRF-CR on end-products of methionine metabolism in liver and serum performed during the daytime (ZT4) (n = 3). Data are presented as mean values ± SEM.