Quinidine

Wild-type cells or cells transgenically expressing a translational fusion of CCA1 to luciferase from the CCA1 promoter (CCA1-LUC) 10 (link) were grown under 12h/12h light/dark cycles in artificial sea water (24 g/l NaCl, 4 g/l Na₂SO₄, 0.68 g/l KCl, 200 mg/l NaHCO₃, 100 mg/l KBr, 25 mg/l H₃BO₃, 3 mg/l NaF, plus hydrous salts: 50 mM MgCl₂*6H₂O, 10 mM CaCl₂*2H₂O, 0.1 µM SrCl*6H₂0), supplemented with Guillard's F/2 marine enrichment solution and 10 nM H₂SeO₃. Full medium was adjusted to a salinity of 30 ppt.
Imaging and analysis of luminescent rhythms was performed as described31 (link)–33 . For resetting experiments, magnesium-free media were removed with a multichannel pipette and replaced with magnesium-containing media. In all luminescent imaging experiments, 8 replicate wells constitute n=8, and presented experiments are representative of 3 or more replicate experiments.
For ICP-MS analyses, 30 ml culture was pelleted, washed three times in 1 M Sorbitol to remove sea water, and digested in 100 µl nitric acid (69%, ARISTAR grade, VWR International) spiked with 345 ppb indium (VWR International) at RT for ~3 hours. Samples were then diluted to a final concentration of 2% v/v nitric acid and 10 ppb Indium prior to analysis on an Agilent 7500ce with octopole reaction system. Serial dilutions of ICP-Multi-element solution IV (Merck, Certipur) was used for calibration of all the metals analysed and to check for instrument drift. A standard reference material SRM1643e (NIST) was analysed to validate the calibration. Indium was used to correct for dilution errors introduced during handling. ICP-MS data reported is based on three replicate flasks, each sampled every timepoint (n=3). Results presented have been verified in a replicate experiment, and outliers were excluded if they were >2 S.D. from the mean.
Cell extracts for luminescent Mg²⁺ and ATP assays were made from 3 replicates (n=3) of 5 ml cell culture, pelleted and washed with 1 M Sorbitol, and resuspended in 100 µl medium before adding 100 µl 2x extraction buffer (1% Triton X-100, 300 mM NaCl, 100 mM HEPES). 25 µl of extract was boiled and added to 75 µl of assay mix (40 mM HEPES, 1 mM luciferin, 0.05 mg/ml QuantiLum (Promega), and either 1 mM MgCl₂ or 10 µM ATP). Luminescence was measured on a TopCount (Packard) plate reader against a standard curve. As Mg²⁺ ions that remain tightly bound to cellular macromolecules such as membrane components and DNA are not detected by this alternative assay, the relative amplitude of [Mg²⁺]_i changes observed using this assay were substantially larger than measured by ICP-MS. Quinidine, CHA and CPA were made up in medium and added 24 hours prior to cell lysis for chronic treatments. Results were verified in one or more replicate experiments. For puromycin experiments (Fig. 4), cobalt ammines or vehicle were added at ZT6 or ZT18, and 0.5 mg/ml puromycin was added 20 minutes before harvesting cells at ZT11 and ZT23 (required concentration and incubation time determined empirically to reduce expression from a constitutive promoter driving luciferase by ~half). Analysis of incorporation was performed as described for U2OS cells below. Loading control was RbcL (Coomassie).
To identify potential Ostreococcus transporter proteins, mammalian sequences for all classes of SLC and all known magnesium transporters were blasted onto the Ostreococcus proteome using DELTA-BLAST (NCBI), and gene models were then taken from the latest version of the Ostreococcus genome 34 (link) using the Orcae service 35 (link) (Gent University).