CK1δ inhibition disrupts the PAC-2 cell circadian clock.

Dose-response of PF-670462, a pan-CK1δ/ε inhibitor and PF-4800567, a selective inhibitor of CK1ε, in the zebrafish PAC-2 cell line. Bioluminescence assay of cells transfected with per1b:Luc (A and C) or Ebox:Luc (B and D), and treated with PF-670462 (A and B) or with PF-4800567 (C and D) at the indicated time (arrow). Cells were maintained under LD cycles, and then the inhibitor was added to the cell culture 1.5 h before lights on (black arrows) at different concentrations (PF-670462: 0.5 μM- light blue line, 1 μM- blue line, 2 μM-green line, 5 μM- red line. PF-4800567: 5 μM- green line, 7.5 μM- light blue line and 10 μM- blue line). Controls were treated with DMSO (black line) or with culture medium (gray line). After three LD cycles cell were transferred to DD. Bioluminescence is plotted on the y-axis and time (hours) on the x-axis. White/black bars show the light and dark periods, respectively. Clock-controlled rhythmic promoter was disrupted by PF-67046 but not by PF-4800567, thus CK1δ activity appears to be important for peripheral circadian clock function.

本研究旨在探讨PF-670462，一种广谱CK1δ/ε抑制剂，以及PF-4800567，一种选择性CK1ε抑制剂，在斑马鱼PAC-2细胞系中的剂量反应关系。通过生物发光实验对过表达per1b:Luc（A和C图）或Ebox:Luc（B和D图）的细胞进行检测，并分别用PF-670462（A和B图）或PF-4800567（C和D图）处理，于指定时间（箭头所示）进行。细胞在光照周期下维持生长，并在光照前1.5小时加入抑制剂（黑色箭头），不同浓度下进行（PF-670462：0.5 μM-浅蓝色线，1 μM-蓝色线，2 μM-绿色线，5 μM-红色线；PF-4800567：5 μM-绿色线，7.5 μM-浅蓝色线，10 μM-蓝色线）。对照组用DMSO（黑色线）或培养基（灰色线）处理。经过三个光照周期后，细胞转移至暗周期。生物发光值以y轴表示，时间（小时）以x轴表示。白色/黑色条表示光暗周期。PF-670462破坏了时钟控制的启动子，而PF-4800567则未影响，因此CK1δ活性似乎对于外围昼夜节律时钟功能至关重要。