Fig. 1. CoCl₂ alters circadian gene expression in synchronized fibroblasts. mRNA levels of Stra13 (A), Dec2 (B), Rev-erbα (C), Bmal1 (D) and Dbp (E) were determined using real time PCR in serum shocked NIH-3T3 cells grown in the absence (empty circles) or presence (filled triangles) of 200 μM CoCl₂. For all genes, the constitutively expressed 36B4 mRNA was used for normalization. For each time point the mean ± SEM of three independent experiments is shown. Circadian period (h) was statistically tested for all genes under the control and CoCl₂ conditions using the cosinor analysis, and are indicated in panel (F) (NA, not applicable).

Fig. 2. STRA13 and DEC2 negatively regulate the Rev-erbα and Dbp promoters. NIH-3T3 cells were transfected with either the pRE-Δtk-Luc (A), P1Rev-erbα-Luc (A, B) or pGL3+857-Luc (C) reporter plasmid and the indicated combination of expression plasmids. The control condition (white bar) in which an empty expression plasmid was used was given the arbitrary value of 1. Data are expressed as mean ± SEM from three independent experiments.

Fig. 3. Alteration of circadian gene expression in hypoxic tumours. (A) Western blotting analysis of HIF1α protein expression in normoxic and CoCl2 treated NIH-3T3 cells, and in GOS tumour. The HIF1α protein is indicated by an arrow and * denote non specific bands. (B) Expression of Stra13, Dec2, Rev-erbα, Bmal1, and Dbp mRNA determined using real time PCR in GOS transplanted tumours and in bone from untransplanted animals. The liver was analyzed as a control tissue for the transplanted animals. For all genes, the constitutively expressed 36B4 mRNA was used for normalization. Values were expressed relative to the lowest level that was given the arbitrary value of 1. The significance and acrophase value determined by the cosinor analysis are indicated for each gene in the three tissues. Values are mean ± SEM from 3 to 4 animals. The white and black bars represent the light and dark phase, respectively.