Fig. 5

p20 regulates circadian cell-cycle timing by inhibiting the G1/S transition. (A) Yeast two-hybrid analysis of p21 and p20 interactions with CDK1, CDK2, and PCNA. Four independent transformants from each plasmid combination were selected on Leu/Trp media. Protein interactions were assayed on ?His, His/+10 mM 3-AT, and ?Ade media. (B) Representative histograms of DNA content from embryos at 70?80% epiboly injected with 60 pg/embryo of HA or HA-p21 mRNA at the single-cell stage (Left). Cell-cycle distribution of the same embryo samples determined by the Watson Pragmatic Model (Right). (C) Representative histograms of DNA content from embryos at 70?80% epiboly injected with 40 pg/embryo of Flag or Flag-p20 mRNA at the single-cell stage (Left). Cell-cycle distribution of the same embryo samples determined by the Watson Pragmatic Model (Right). **P < 0.01, ***P < 0.001. qPCR analysis of p21 and p20 expression plotted against the percentage of cells in S phase in PAC2 cells (D) and in 6-d-old larvae (E). (F) Quantification of BrdU-positive cells present in cross-sections of the hindbrain and the intestinal bulb of 6-d-old larvae. For each time point, three independent larvae were analyzed for a total of 12 sections. White and gray backgrounds represent light and dark phases, respectively.