The orphan receptor Rev-erb gene is a target of the circadian clock pacemaker

Supplementary figures

Supplementary figures

Files in this Data Supplement:

• Supplementary figure 1 - P1 and P2 start sites characterized by RNase protection assays in rat osteosarcoma ROS cells. (A and B) lane 1, probes; lane 2, tRNA; lane 3, ROS mRNA; lane 4, molecular weight marker. (A) P1 probe is 736 bp antisense mRNA probe overlapping the 5’ E1A exon. The P1 major start site is indicated by a black circle and the minor one by a diamond. Nucleotide sequences below correspond to the 5' of the two protected regions and arrows point out the bases mapped for P1 rat start sites. (B) P2 probe is 650 bp antisense mRNA probe overlapping the 5' E1B exon. The start site is indicated by a black circle. Nucleotide sequence below is the mapped region and the arrow points out the start site.
• Supplementary figure 2 - (A) Circadian analysis of REV-ERBα by western blot in Rat-1 cells nuclear extracts. Nuclear extracts were prepared after a 2 hours serum-shock from time 0 to 24. REVERBα isoforms were detected using a rabbit polyclonal serum directed against the REV-ERBα LBD (from Preitner) and an ECL detection Kit (Amersham). Only the REV-ERBα1 isoform is observed. (B) Top: EMSA experiment using the circadian Rat-1 nuclear extracts on a2 E-box versus mutated one (a2*). Lanes 1 to 3 : nuclear extracts from STO cells transfected with vectors expressing CLOCK and BMAL1 were used as controls (CB extracts). Binding of endogenous CLOCK-BMAL1 was assayed on a2 E-box (lanes 4 to 9) or mutated a2 E-box (lanes 10 to 16) probes using the nuclear extracts prepared after a 2 hours serum-shock. No binding was detected on mutated a2 neither using circadian nuclear extracts nor CB extracts. (Bottom) Relative quantitation of the circadian specific binding on a2 E-box probe corrected by a normalization with a Bradford dosage. (C) Top: EMSA experiment, as described in B, on mPer1 E-box and mutated Per1(*) probes. Bottom: Relative quantitation of the circadian specific binding on Per1 E-box probe.
• Supplementary figure 3 - (A) Basal activities of P1+P2, P1 and P2 promoters checked by transient transfection assays in Cos-1 and Ros17.2/8 cells using 200 ng of plasmid. Luciferase assay mesures were done after 48 h. (B) Transient transfection assays were performed to check the basal effect of each effector: neutral, CLOCK, BMAL1, CRY1. 40 ng of promoter reporters were added with 150 ng of effectors (in PCDNA3) and 150 ng of empty PCDNA3 plasmid. For CLOCK-BMAL1 activation each plasmids were mixed in an equal quantity to reach 300 ng. Luciferase assay mesures were done after 72 h.