Figure 5: MYC inversely regulates the circadian clock and proliferation.

(a) MYC:V5 WT and V393D U2OS cells were stained with propidium iodide 48 h after induction and DNA content was quantified by FACS (n=3). Values indicate difference (in %) to PBS-treated cells in the respective cell cycle phase. (b) Left panel: fluorescence microscopy of U2OS t-rex tetO-MYC:V5 cells stably expressing mCherry-Cdt1 (FUCCI-Red G1 marker) 48 h after treatment with doxycycline to induce MYC:V5 (MYC ox) or PBS (Ctrl). Scale bar, 300 μm. Right panel: quantification of cells in G0 or G1 phase by mCherry-Cdt1 expression (n=3) (c) FACS analysis of U2OS cells stained with propidium iodide 48 h after transfection with MYC siRNA (n=3). Values indicate difference to cells transfected with negative siRNA. (d) Growth curve of U2OS cells transfected with MYC siRNA and negative siRNA (n=3). (e) Western blot analysis (left) and densitometric protein quantification (right) showing that MYC depletion by siRNA supports increased BMAL1 and CLOCK expression in U2OS cells (n=3). (f) Relative amplitudes (ChronoStar software) of circadian luciferase rhythms of indicated U2OS reporter cell lines (n=3). The cells were transfected with MYC siRNA and negative siRNA as indicated. Data are presented as mean±s.e.m. *P<0.05; Student’s t-test (e,f), one-way (b,c) and two-way (a,d) analysis of variance (ANOVA) with Bonferroni post-test.