Figure 4

Accumulation of REV-ERBα and CLOCK does not show obvious circadian rhythmicity. (A) A mixture of equal amounts of protein samples collected from U2OS-P_Bmal1::Fluc/ΔBmal1/P_TRE3Gs::Myc-Bmal1 strain-2 cell cultures from 0 to 52 h after the addition of 100 nM dexamethasone in the presence of each doxycycline (DOX) concentration was subjected to immunoblot analysis using anti-REV-ERBα antibodies. The amount of protein was calculated by the density of each band versus total protein and were normalized using the value from the 1 µg/mL DOX group. The results are shown as mean ± SEM (N = 3). Different characters (a, b) indicate significant differences (one-way ANOVA followed by Tukey’s multiple comparison test, P < 0.05). (B–E) Time course of protein expression in the presence of 0.1 µg/mL (B,D) and 1 µg/mL DOX (C,E). Images showing the protein bands detected for REV-ERBα (B–C), CLOCK (D–E), and total protein (TP) stains (upper panels). Asterisks in (B,C,E) indicate nonspecific bands. Markers (filled traingle, filled diamond, and filled circle) indicate three biological replicates. For REV-ERBα detection, the same blots used for MYC-BMAL1 detection were reprobed. The protein amount was quantified using densitometry (lower panel). The relative expression of REV-ERBα (B–C) and CLOCK (D–E) protein was calculated by the density of each band vs. total protein and were normalized against the intensity of pooled 0 to 52 h samples. Black lines indicate the average values of three biological replicates. No significant rhythmicity was detected for REV-ERBα at 0.1 and 1 µg/mL DOX (JTK cycle test, ADJ.P = 1) or CLOCK at 0.1 µg/mL DOX (JTK cycle test, ADJ.P = 0.77). CLOCK at 1.0 µg/mL DOX exhibited significant rhythmicity (JTK cycle test, ADJ.P = 0.029) with low amplitude (JTK cycle test, AMP = 0.23).