Files in this Data Supplement:

• Supplemental Methods and Tables - (Microsoft Word) (54 kb)
• Figure I - (EPS) (325 kb) Platelet reactivity does not display diurnal variation. (A) Fold over basal dose response curves for platelet P-selectin expression in response to the PAR4 agonist peptide AYPGKF. Aliquots of whole blood harvested from WT mice at ZT2, ZT8, or ZT14 were left unstimulated or stimulated with increasing amounts of AYPGKF, and analyzed for P-selectin expression by flow cytometry. Ratios of stimulated to unstimulated mean fluorescent intensity (fold over basal) were calculated. LogEC50 was statistically indistinguishable between all data sets (n=6). Dose response curves for WT whole blood platelet aggregation, as measured by impedence in response to collagen (B), IBOP (C), or U46619 (D). Aliquots of whole blood harvested from WT mice at ZT2, ZT8, or ZT14 were stimulated with increasing amounts of collagen. LogEC50 was statistically indistinguishable between all data sets (n=5-13).
• Figure II - (EPS) (2.64 MB) Endothelial cells express clock proteins but do not display transcript oscillations in response to the serum shock model. (A) Western blot analysis of whole cell lysate from hCAEC or control (HeLa) showing expression of core clock proteins PER2, CLOCK, BMAL1, and NPAS2. Blots are representative of three experiments. (B) Quantitative analysis by real-time PCR of hPer2 RNA expression in hCAECs treated with 50% serum (serum shock) or 0.3% serum (media change control) for 2hrs starting at T0. Each value is normalized to h36b4 and expressed relative to the mean of each experiment. Values are meanąSEM (serum shock n=4, media change n=3) (P=0.034 for serum shock; P=0.015 for media change). * P<0.05 for serum shock T0 vs. T2 only. No other timepoints significantly different. Quantitative analysis of hPer1 (C), hPer2 (D), hCry1 (E), and hBmal1 (F) RNA expression in HUVECs under the same conditions. Values are meanąSEM (serum shock, n=4) or meanąSD (media change, n=2). (P=0.002 and 0.054 for Per2 serum shock and media change, respectively; P=0.008 and 0.052 for Bmal1serum shock and media change, respectively; P=0.408 and 0.176 for Per1serum shock and media change, respectively; P=0.107 and 0.430 for Cry1serum shock and media change, respectively). *P<0.05 for serum shock T0 vs. T4. No other timepoints significantly different.
• FIgure II - (EPS) (949 kb) Specificity of Cre^Tek-mediated excision of the Bmal1^fx allele. (A) Specificity of Bmal1^fx excision by Cre^Tek was determined by genotyping for both the unexcised and excised alleles of Bmal1^fx in genomic DNA from various tissues obtained from Bmal1^fx/fx and Bmal1^fx/fx-Cre^Tek mice, as previously described1. LIV, liver; AOR, aorta; KID, kidney; HRT, heart; BM, bone marrow. Real-time PCR analysis of endothelial cell (EC) or smooth muscle cell (SMC) mRNA isolated from Bmal1^fx/fx or Bmal1^fx/fxCre^Tek mice (n=4 all groups, expressed as relative %). (B) Circadian genes mBmal1, mClif, mNpas2, and mPer2. * P<0.05 for Bmal1^fx/fx vs. Bmal1^fx/fxCre^Tek. (C) Output genes mPai-1 and mThbd. (D) Real-time PCR analysis of aortas harvested from Bmal1^fx/fxCre^Tek mice and their Bmal1^fx/fx littermate controls at 4-hr intervals from CT18-CT34. mPai-1 displayed significant rhythmic expression in Bmal1^fx/fx and Bmal1^fx/fxCre^Tek mice (P=0.019), with a significant increase in expression at CT30 in Bmal1^fx/fxCre^Tek (* P<0.05). Rhythmic expression was not observed for either mClif (P=0.422, Bmal1^fx/fx; P=0.518 Bmal1^fx/fxCre^Tek) or mThbd (P=0.150, Bmal1^fx/fx; P=0.413 Bmal1^fx/fxCre^Tek), with no difference between genotypes.
• Figure IV - (EPS) (604 kb) Active plasma PAI-1 and the effect of circadian clock components CLOCK and NPAS2 on Pai-1 mRNA. (A) WT mean active plasma PAI-1 (P= 0.0004, n=5-9). * P<0.01 for ZT2 vs. ZT8 or ZT14. (B) Mean mPai-1 mRNA relative to mGapdh (P<0.0001, n=7-9). * P<0.05 for ZT2 vs. ZT14 within genotypes; †P<0.01 and ‡P<0.001 for comparisons across genotypes. (C) Relative mPai-1mRNA expression in the liver (P<0.0001, n=7-9). * P<0.05 for WT ZT2 vs. ZT8; †P<0.05, ‡P<0.01, §P<0.001 for comparisons across genotypes. Data shown are mean±SEM. ND = not detected.