Lumicycle analysis

Methods used were similar to those described previously (Nakamura et al., 2011 (link)). Mouse SCN slices were made with the same method described earlier. At ZT 4, the medium change was performed to wash drugs. Explants were transferred on to Millicell membranes (0.4 µm, PICMORG50, Merck KGaA) resting on 1.2 ml of recording media that contained freshly added 0.1 mM luciferin (sodium salt monohydrate, Biosynth Chemistry & Biology, Itasca, IL), and the 35 mm dishes were sealed using autoclaved high-vacuum grease (Dow Corning, Midland, Michigan). Tissue explants were inserted in the LumiCycle photomultiplier tube (Actimetrics, Wilmette, IL), and bioluminescence was monitored at 37℃. The bioluminescence signal was counted in 1 min bins for every 10 min for at least 5 days without changing media. The data were normalized by subtraction of the 24 h running average from the raw data and then smoothed with a 2 h running average (LumiCycle analysis, Actimetrics).

1.5 × 10⁵ U2OS PER2-Luc (U2OS-D15) or U2OS BMAL1-Luc (U2OS-C26) cells were inoculated into 35 mm culture plates (Costar, #9102) in triplicate. On the next day, cells in each well were synchronized with 2 h treatment of 100 nM dexamethasone and recorded in a Lumicycle as described previously [31 (link)]. Different concentrations of lithium chloride (LiCl) were added at peaks of the luminescence oscillation, using sodium chloride (NaCl) as the vehicle control. Bioluminescence data were analysed with the Lumicycle analysis program (Actimetrics, USA) to obtain circadian parameters such as period and amplitude.

A stable U2OS-B6 cell line that expresses a destabilized firefly luciferase gene under the control of the mBmal1 promoter was obtained from Dr Satchidananda Panda (Vollmers et al., 2008 (link)). siRNAs targeted to LBR, LMNB1, MAN1, or SMAD1 (10 nM, Invitrogen; Carlsbad, CA; see Supplementary file 1A) were individually transfected into 35-mm culture dishes using Lipofectamine RNAiMAX (Invitrogen). For overexpression of FLAG-tagged constructs, plasmid (2 µg) was distributed into each well along with FuGENE HD (4 μl, Roche; Switzerland). For co-transfection of MAN1 siRNA and Bmal1, Lipofectamine 2000 (Invitrogen) was used. 24 hr after transfection, cells were synchronized with 100 nM dexamethasone in serum-free DMEM containing 10 mM HEPES (pH 7.5) at 37°C for 2 hr. Following synchronization, the media were replaced with phenol red-free DMEM supplemented with 10 mM HEPES and 40 µM Luciferin-EF (Promega). Cells sealed with coverslips were incubated in a 32-channel LumiCycle (Actimetrics; Evanston, IL) to monitor real-time bioluminescence for 5 days. Data were analyzed using LumiCycle Analysis (Actimetrics).