Supplementary Figure 1: Heme oxygenase 1 is regulated by the circadian clock: additional data.

(a) Ho-1 mRNA levels oscillate in mouse peritoneal macrophages. These data are taken from our experiments published in Keller, M. et al. Proc. Natl. Acad. Sci. USA. 106, 21407-21412 (2009). Cells harvested every 4 h via peritoneal lavage from four C57BL/6 mice kept in constant darkness were magnetically purified for CD11b surface expression. Three individual RNA samples of each time were pooled and subjected to global gene transcription measurement by using Affymetrix microarrays. Circadian oscillation of Ho-1 transcript is significant (p < 0.005 CircWave). (b) Ho-1 mRNA levels do not oscillate in primary hepatocytes from Bmal1^-/- mice. Ho-1 mRNA levels in hemin and CoPPIX treated (30 μM each) primary hepatocytes from Bmal1^-/- mice. Cells were dexamethasone-synchronized and harvested after 24 hour and 40 hours, which correspond to trough and peak of Ho-1 mRNA levels in wild-type cells (see Fig. 2b). Data are normalized to Gapdh expression and presented relative to mean expression in untreated cells. Given are means ± sd of three independent samples. (c) Conserved E-box (blue) in mouse, rat and human Ho-1 promoters close the transcription start site (yellow) and start of coding region (red). (d) Ho-1 mRNA levels are reduced in unsynchronized primary hepatocytes from Bmal1^-/- mice compared to hepatocytes from wild-type littermates (wt). Data are normalized to Gapdh expression and presented relative to mean expression in wild-type cells. Given is mean ± sd of three independent samples.