Figure 9.

Rhythmic expression of Clock and Nrf2 antioxidant genes at transcriptional and translational level in C57BL/6 mice in vivo and ROS levels under 12L:12D condition. (A) Regulation of clock-controlled antioxidants mRNA expression in eye lenses at different Zeitgeber times (ZT). C57BL/6 mouse eye lens were harvested at 4 h intervals over the course of 48-h period, as indicated, and transcript levels were analyzed by using RT-qPCR. Bmal1 and antioxidants of Nrf2/ARE-mediated genes mRNA expression were examined at the indicated ZT. The data represent the mean ± S.D. from three independent experiments. (B) Circadian oscillation of clock and Nrf2 antioxidant targets protein, Bmal1, Clock, Nrf2, Prdx6, NQO1, and HO1 in vivo. C57BL/6 mouse eye lens were harvested at 4 h intervals over the course of 24 h, as indicated, and total protein were isolated and equal amount of protein were immunoblotted with antibodies as indicated. Circadian profiles for Bmal1, Clock, Nrf2, Prdx6, NQO1, and HO1 assayed by immunoblotting from C57BL/6 mouse eye lens. Protein blots were quantified using densitometer, and levels were normalized to corresponding β-actin and tubulin levels; lined graph below shows the protein bands. (C) Circadian clock controls of ROS at ZT interval. C57BL/6 mouse eye lens were harvested at 4-h intervals over the course of 24 h, as indicated, and lens homogenate prepared as described in Materials and Methods. ROS levels were measured using fluorescent dye H2-DCF-DA dye methods. Data represent means ± S.D. of three independent experiments.