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. 2015 Oct 2;10(10):e0139725. doi: 10.1371/journal.pone.0139725

Fig 6. Impact of 585KA mutation on circadian oscillations of CRY1 protein, Bmal1 promoter activity as well as clock output gene.

Fig 6

(A) Circadian oscillations of CRY1-WT vs. CRY1-585KA in Hepa1 cells. 24 hr after transfection with Cry1 expression vectors (WT vs. 585KA), Hepa1 cells were synchronized by serum shock and harvested every 8 hr between 16 and 64 hr. The protein levels of CRY1 protein (WT vs. 585KA), BMAL1, and CLOCK were determined by immunoblotting with anti-FLAG or with protein-specific antibodies. (B) Impact of CRY1 WT or CRY585KA on oscillations of Bmal1-luc in U2OS cells. The Bmal1-luc U2OS stable cells were transfected with GFP, Cry1-WT, or Cry1-585KA and then subjected to Lumicycle analysis. The period length and amplitude values were calculated and compared. *p-value < 0.05 between CRY1-WT and Cry1-585KA group by the student’s t-test (n = 6). (C) Increased amplitude of the endogenous clock gene oscillations in cells overexpressing CRY1-585KA. U2OS cells were transfected with expression vectors encoding either Cry1-WT or Cry1-585KA. 24 hr post-transfection, cells were synchronized by serum shock and collected at the indicated time points. The endogenous Dbp mRNA levels were measured by RT-qPCR. The data were plotted as Mean ± S.D. (n = 4). The AUC (area under curve) analysis for Dbp mRNA oscillations was presented as well. * p-value < 0.05 by Student’s t-test.