Figure 2: REV-ERBα mRNA is induced by MYC regardless of synchronization method and in multiple cell line and tumor models.

(a,b) MYC was induced in U2OS MYC-ER cells that were synchronized with dexamethasone in two different schemes. ‘Post-Shock’¹: as in Fig. 1, cells were treated±4 OHT (with ethanol serving as a loading control for all experiments) for 24 h. The ‘24’ h sample was collected, then 0.1 μM dexamethasone was added to media and the ‘48’ h sample was collected 24 h later. ‘Pre-Shock’²: 1 μM dexamethasone was added for 20 min, then cells were washed once in PBS and fresh media was added±4OHT. Cells were collected at the indicated times after media change. (a) mRNA was collected at the indicated times, and endogenous REV-ERBα (NR1D1), BMAL1 (ARNTL), PER2, and ODC1 were determined by RT-PCR, normalized to β2M. mRNA (FC)=fold change. Data are averages of biological triplicates with error bars representing s.d., and *P<0.05 by Student’s t-test of 4 OHT (MYC-ON) samples relative to ethanol (MYC-OFF) samples at each time point. (b) Samples from (a) were processed for protein expression of REV-ERBα, BMAL1 and PER2. Tubulin serves as a loading control. Molecular weights are noted in kDa. (c) Previously published RNA-Seq data¹³ from U2OS cells expressing exogenous MYC under the control of a TET-ON system and treated±1 μg ml⁻¹ doxycycline for 30 h. REV-ERBα (NR1D1), BMAL1 (ARNTL), PER2 and ODC1 were determined. Data are presented as linear fold change (FC) and represent biological triplicates, and *P<0.05 of MYC-ON samples relative to MYC-OFF samples as previously described¹³; NS, not significant. (d) Previously published RNA-seq data¹⁴ from liver tumors driven by a MYC-TET-OFF system. Mice were fed water or doxycycline for 16 h to turn off MYC. Rev-erbα (Nr1d1), Bmal1 (Arntl), Per2 and Odc1 were determined. Data are presented as linear FC and represent biological duplicates, and *P<0.05 of MYC-ON samples relative to MYC-OFF samples as previously described¹⁴.