Lumicycle data analysis program

Ex vivo circadian bioluminescence measurement was performed as previously described (Yoo et al., 2004). Briefly, hearts were quickly removed and placed in warm PBS solution to remove blood. Left and right atrial tissues was manually dissected to small pieces of equivalent size (four pieces per atrium); both right and left atria were used. The dissected atrial tissues were cultured on Millicell culture membranes (PICMORG50, Millipore) in 35 mm dishes containing 2 mL DMEM media (Invitrogen) supplemented with 352.5 μg/mL sodium bicarbonate, 10 mM HEPES (Invitrogen), 2 mM l-Glutamine, 5% FBS, 25 units/mL penicillin, 25 μg/mL streptomycin (Invitrogen), and 0.1 mM luciferin (l-8240, Biosynth AG). DMSO or moricizine (30 µM) was added to the recording media. Bioluminescence was recorded continuously using the LumiCycle luminometer (Actimetrics). Data were analyzed using LumiCycle data analysis program (Actimetrics).

To monitor circadian rhythms in MDA-MB-231 and MDA-MB-468 cells, we transfected and generated clones with stable expression of the Bmal1::Luciferase and Per2::Luciferase reporters under blasticidin selection [42 (link), 43 (link)]. U2OS cells were used as a control cell line to rhythmically express these reporters. Cell lines with stable expression were subjected to real-time bioluminescence monitoring by using LumiCycle 32 (Actimetrics, Wilmette, IL, USA). Cells were cultured on 35 mm plates and were synchronized with 200 nM dexamethasone (Sigma) for 1 h. After DMSO or NOB (10 µM) containing recording media [44 (link)] were added, the dishes were sealed with vacuum silicon grease and bioluminescence was measured in LumiCycle 32 for continuous bioluminescence monitoring over 4 days. The data were detrended using a first-order polynomial, and then best-fit to a sine wave estimated by a Levenberg–Marquardt algorithm for measurement of circadian parameters in the LumiCycle data analysis program (Actimetrics).

Real-time circadian bioluminescence monitoring was conducted by using adult mouse ear fibroblast cells isolated from Per2::LucSV knock-in mice by replacement of the 3′-UTR with an SV40 late poly(A) sequence as described [59 (link)]. Cells were grown to confluency on 35 mm dishes in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin, and subsequently synchronized with 200 nM dexamethasone (Dex) for 1.5 h or forskolin (Fsk, 1 or 5 µM) for 1 h [37 (link),41 (link),59 (link)]. Moricizine (Sigma–Aldrich or Santa Cruz) was then added at concentrations of 0 (vehicle control), 0.3, 1, 3, 10, 20, and 30 µM, or Flecainide (Sigma-Aldrich) at 3, 10, and 30 µM, along with luciferin-containing recording media. The dishes were then tightly sealed with vacuum grease and placed in a LumiCycle luminometer (Actimetrics) for continuous bioluminescence monitoring over six days. The data were detrended using a first-order polynomial, then best-fit to a sine wave estimated by a Levenberg–Marquardt algorithm for measurement of circadian parameters in the LumiCycle data analysis program (Actimetrics).