Extended Data Fig. 3: Effect of entry into S-phase on 2CLC reprogramming.

a. ESCs were synchronized using a CDK1 inhibitor, after which existing 2CLCs were removed. Cells were subsequently grown with or without CDK1 inhibitor and newly emerging 2CLCs were quantified hourly by FACS until 6 h after block release. b. ESCs were synchronized using a PLK1 inhibitor, after which existing 2CLCs were removed. Cells were subsequently grown with or without PLK1 inhibitor and newly emerging 2CLCs were quantified by FACS 6 h after block release. c. Cell cycle profiles determined by FACS for propidium iodide staining of ESCs after release from PLK1 inhibitor, which corresponds to Extended Data Fig. 2b. d. Cell cycle profiles based on propidium iodide content of ESCs after release from CDK1 inhibitor, which corresponds to Extended Data Fig. 2a. e. Cell cycle profiles based on propidium iodide content of ESCs after collection in G1-phase using FACS without the addition of drugs (for example without cell cycle synchronization) based on the FUCCI reporter. These data correspond to Fig. 2e. f. The panel shows the data from the double thymidine block and release experiment described in Fig. 2A and the corresponding fit. The estimation of f_s obtained from this fit was then used to identify the values of \(\frac{{f_{G1}}}{{f_S}}\) and \(\frac{{f_{G2M}}}{{f_S}}\) compatible with the data, shown in Fig. 2f. These values lie on a line, shown in Fig. 2f, computed from eq. (1) in the Methods. The values of the parameters used are: \(\frac{{N_{2c}}}{{N_E}} = 0.01,T_c = 8\;h,\) \(\frac{1}{{\omega - \varphi _{2c}}} = 12\;h.\) In a and b, the bar plots show mean±S.D. and dots indicate the values of each replicate. n indicates the number of independent biological replicates. Statistical comparisons were performed by two-sided Student’s t-test.