As shown in Figure e, this revealed that the length of G2+M+G1 (TC–TS), as indicated by the time point at which the EdU labelling index reached the plateau, was longer for the total population of BPs (23.3 h) than APs (14.1 h). In contrast, the proportion of the cell cycle comprising S-phase, as indicated by the intercept of the cumulative EdU labelling curve with the y axis, was smaller for BPs (12%) than for APs (26%). The growth fraction was nearly 100% for both, APs and BPs. Calculation of the length of S-phase (TS) and the total cell cycle (TC)24 yielded values of 5.0 and 19.1 h for APs and 3.2 and 26.5 h for BPs. The present TC data determined by cumulative EdU labelling are very similar to those determined by live imaging in organotypic slice culture7,20.
Determining the proportion of NPCs in S-phase using an independent method, that is, proliferating cell nuclear antigen (PCNA) immunostaining. Cell nuclei in S-phase typically exhibit a punctate pattern of PCNA immunoreactivity, which reflects sites of DNA replication, whereas nuclei in G1 and G2 show diffuse PCNA immunoreactivity
Analysis of the appearance of EdU label in mitotic figures (mitotic EdU labelling index) (in another word, all ph3 positive cells are reaching 100% Edu label plateau) revealed a very similar time course for the four NPC populations (Fig. 4e), with almost all mitotic NPCs becoming EdU+ 2–3 h after EdU administration, indicating an average G2-length (TG2) of <2 h (∼1.6 h). With regard to M-phase, we first determined the percentages of total Tis21-GFP− and Tis21-GFP+ APs and BPs, identified as in Figure 1, that were in M-phase by DAPI staining and PH3 immunofluorescence as in Figure 4a–d, and then calculated the length of M-phase (TM) in the four NPC populations from the respective TC. This revealed that TMwas longer in APs than in BPs, being longest in Tis21-GFP− APs (Table 1).
