Fig. 2: MYCN directly binds to clock gene promoters and downregulates the positive arm of the clock.
From: Restoration of the molecular clock is tumor suppressive in neuroblastoma
a Left panel: MYCN protein expression in MYCN3 (MYCN Tet-ON) and LAN5 ShMYCN cells after DOX (1 µg/ml) induction for 48 and 72 h, respectively. Protein expression was analyzed by densitometry (Image J v1.42q). CypB served as a loading control and MYCN/CypB ratio was determined (n = 2 independent experiments). Right panel: mRNA levels of MYCN, REV-ERBα, RORα, and BMAL1 in MYCN3 (MYCN Tet-ON) and LAN5 ShMYCN cells following DOX induction for 48 and 72 h, respectively. Data are mean ± SD (n = 3; ****p < 0.0001; two-tailed unpaired t-test). b mRNA and protein expression of REV-ERBα, RORα, and BMAL1 in SK-N-AS MYCN-ER cells with and without 4OHT (1 µM) for 0, 8, 16, 24, and 48 h. mRNA data are mean ± SD (n = 3; ****p < 0.0001; two-tailed unpaired t-test). Protein expression data were analyzed by densitometry (Image J v1.42q). CypB served as loading control and REV-ERBα/RORα/BMAL1/CypB ratios were determined (n = 2). REV-ERBα and RORα were run on the same gel. c MYCN ChIP-qPCR assays in LAN5 and Tet-21/N (MYCN Tet-OFF) cells following tetracycline treatment (2 μg/ml for 48 h). Input (white bars) and MYCN IP (blue bars) samples were analyzed by q-PCR using specific primers for REV-ERBα, RORα, BMAL1, and RORc (Supplementary Table 3). Data are mean ± SD (n = 3; ****p < 0.0001; two-way ANOVA with Dunnett’s multiple comparisons test). FC fold change, FE fold enrichment.
