Supplementary Figure 5: Heme oxygenase–derived CO is essential for normal circadian dynamics in mammalian cells: additional data.
From: Reciprocal regulation of carbon monoxide metabolism and the circadian clock
(a) Circadian dynamics of synchronized primary fibroblasts from Ho-1-/- mice lentivirally transduced with (i) shRNA constructs targeting Ho-2 or a non-silencing (ns) control and (ii) a Bmal1 promoter-luciferase reporter construct. Shown are representative examples of raw data time-series. For detrended time series and period quantification see Fig. 5a. Note, that the absolute light levels decrease upon Ho‑2 knockdown consistent with the overall increase in Rev-Erbα levels and thus putatively lower Bmal1 transcript levels (compare Fig. 5b). (b) CO partly rescues the long period oscillations in heme oxygenase depleted cells. Circadian oscillation dynamics of synchronized primary fibroblasts from Ho-1-/- mice lentivirally transduced with (i) shRNA constructs targeting Ho-2 and (ii) a Bmal1 promoter-luciferase reporter construct. Cells were continuously treated with 6% CO or N2. Shown are representative examples of raw data time-series. For detrended time series see Fig. 5c. (c) Exogenous CO treatment has no effect on oscillation dynamics of wild-type cells. Circadian oscillation dynamics of synchronized primary fibroblasts from wild-type mice lentivirally transduced with a Bmal1 promoter-luciferase reporter construct, which were continuously treated with 6% CO or N2. Shown is a representative example of raw data (upper panel) and detrended time-series (lower panel). Note, since CO can only shorten the circadian period in Ho-depleted cells (Fig. 5c) but not in wild-type cells, period lengthening in Ho-depleted cells is very likely specifically due to the lack of endogenous CO rather than to other functions of heme oxygenases.
