Fig. 9: TRF normalizes protein endogenous circadian rhythms in breast tumors.

a Western blot analysis of the clock protein BMAL1 and phospho-BMAL1(Ser42) in tumor samples collected at different time points (ZT0, 4, 8, 12, 16, 20) over a 24 h period in mice on ad libitum HFD (AL—orange), time-restricted HFD (TRF—green), or chow diet (NC—blue). ZT0 indicates 6 am and the start of the light phase. b Quantification of the normalized phospho-BMAL1(Ser42), total BMAL1, and the phospho-BMAL1/BMAL1 ratio is graphed over 48 h to facilitate visualization of rhythms (n = 4 mice/time/group). Gray rectangles indicate period of lights off. c Western blot analysis of the protein CRY1 in tumor samples collected at different time points (ZT0, 4, 8, 12, 16, 20) over a 24 h period in mice on ad libitum HFD, TRF or NC. d Quantification of the normalized CRY1 protein expression (n = 4 mice/time/group). e Western blot analysis of the clock protein PER1 in tumor samples collected at different time points over a 24 h period in mice on ad libitum HFD, TRF, or NC. f Quantification of the normalized PER1 protein expression (n = 4 mice/time/group). Data are presented as mean ± SEM normalized to β-actin. Twenty-four hour rhythms were analyzed statistically using RAIN with a period of 24 ±4 h, and circadian curves fitted using PRISM using the equation Y = baseline + amplitude*cos(frequency*X + phaseshift) using a frequency of 0.2618 for a 24 h period. Proteins with statistically significant circadian rhythms are shown as curves, acyclic proteins are shown as horizontal lines. Statistical data and circadian parameters are given in Supplementary Table 2.