Figure 3
From: Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations
MYC-BMAL1 protein and mRNA accumulation do not show significant circadian rhythmicity. Total protein and RNA samples were collected from U2OS-PBmal1::Fluc/ΔBmal1/PTRE3Gs::Myc-Bmal1 strain-2 cell cultures every 4 h from 0 to 52 h (for total protein) or 24 to 52 h (for total RNA) after adding 100 nM dexamethasone. (A) Graphs showing relative MYC-BMAL1 protein (left panel) and mRNA accumulation (right panel) after treatment with different concentrations of doxycycline (DOX). For protein accumulation, equal amounts of the samples collected for each DOX concentration were mixed and subjected to immunoblot analysis using anti-Myc antibody. For mRNA accumulation, the average of all the time points for each DOX concentration was calculated. Results were normalized using the average values at 1 µg/mL DOX. Data are shown as the mean ± SEM. N = 3 samples/group, one-way ANOVA followed by Tukey’s multiple comparison test. Different characters (a, b, c) indicate significant differences (P < 0.05). (B,C) Time course of MYC-BMAL1 protein expression after addition of dexamethasone (time 0) in the presence of 0.1 µg/mL (B) and 1 µg/mL DOX (C). Protein samples were collected and subjected to immunoblot analysis in three independent experiments (upper panels). Markers (filled traingle, filled diamond, and filled circle) indicate MYC-BMAL1 bands in three biological replicates, and TP indicates total protein stains. Graphs (lower panels) show the quantification of MYC-BMAL1 amount by densitometry. The intensity of each band was normalized by total protein, and values were normalized using the average of all time points in each series. Black lines indicate the average values of the three biological replicates. No significant rhythmicity in the circadian range (between 20 to 28 h) was detected in the presence of 0.1 and 1 µg/mL DOX (JTK cycle test, ADJ.P = 1). (D) Time course of MYC-BMAL1 mRNA expression after addition of dexamethasone (time 0). Total RNA was extracted from strain-2 cells in the presence of 0.1 and 1 µg/mL DOX and from wild-type U2OS cells. Samples were analyzed by quantitative reverse-transcription PCR. Relative expression was calculated using Pfaffl’s method36 with GAPDH as an internal control. Markers (filled triangle, filled diamond, and filled circle) indicate three biological replicates. Values were normalized using the average of all time points in each series. Black lines indicate the average values of the three biological replicates.
