Figure 4.

Detection of early S phase using EdU. (A) Scheme of experiment. adh1:tk adh1:hENT1 (P2470) cells were arrested in G1 by growth in EMM-N for 16 h at 26°C; cells were released from the cell cycle block by transferring to: (i) EMM only; (ii) EMM containing 12 mM HU; (iii) EMM containing 12 mM HU and 10 μM EdU (all cultures were incubated at 32°C), (B) Analysis of DNA contents of cells from the −EdU, −HU culture showing onset of S phase at around 2 h, (C) Flow cytometric analysis of EdU incorporation in cells from the +EdU+HU culture following conjugation to Alexa Fluor 488 azide, (D) Analysis of DNA contents of cells from the +EdU, +HU culture confirming early S phase arrest. S phase arrest was also shown by the −EdU, +HU culture (Supplementary Figure S5), (E) Cells from (C) were analysed by fluorescence microscopy. Bar = 10 µm and (F) Quantitative analysis of cells shown in (E).