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. 2010 Dec 14;8(12):e1000559. doi: 10.1371/journal.pbio.1000559

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Hirota et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Effect of longdaysin on the circadian period in embryonic fibroblasts from CKIδ deficient mPer2^Luc knockin mice. Luminescence rhythms of CKIδ deficient (CKIδ^Δ2/Δ2) or wild type (CKIδ^+/+) cells were monitored in the presence of various concentrations of longdaysin. Period change relative to the mean of DMSO control of wild type cells was plotted (the mean with SEM, n = 4). (B) Effects of kinase gene siRNAs on the circadian period in Bmal1-dLuc U2OS cells. Luminescence rhythms were monitored after transient transfection with siRNA. Period parameter was obtained by curve fitting, and period change relative to the mean of control was plotted. Data are the mean with variation (n = 2). The period estimation for CSNK1D si4 was not accurate because of poor curve fitting (indicated by asterisk). (C) Dose-dependent effects of kinase gene siRNAs on the circadian period (Ci) and the gene expression (Cii). Luminescence rhythms of Bmal1-dLuc U2OS cells were monitored after transient transfection with various amounts of siRNA (Ci). Representative profiles are indicated in the left panels, and period changes were plotted against siRNA amount in the right panels (the mean with SEM, n = 5–6). Gene expression of unsynchronized cells at time 0 h was analyzed by RT-qPCR (Cii). Expression levels of target gene, homologous gene, and control gene (ACTB) are indicated in the left, middle, and right panels, respectively, by setting control value as 1. Data are the mean with variation (n = 2). (Di) Effect of multiple kinase gene knockdown on the circadian period. Equal amounts of CSNK1D si3, CSNK1A1 si4, MAPK1 si4, and control siRNA were mixed and used for transient transfection. Data are the mean with SEM (n = 8). (Dii) Comparison of single and multiple kinase gene knockdown effects. Effects of single (pink, blue, and purple lines; data from Ci) and multiple (red line; data from Di) gene knockdown were plotted against each siRNA amount. Black line is the theoretical sum of single gene knockdown effects.