Extended Data Figure 9. Function of the m6A methyltransferase complex during pluripotency exit induced by Activin/Nodal inhibition.

(a) qPCR analyses in inducible knockdown (iKD) hESCs cultured in absence (CTR) or presence of tetracycline (TET) for 10 days, then subjected to Activin/Nodal signalling inhibition with SB-431542 (SB) for the indicated time (see Extended Data Fig. 10a). Activin: cells maintained in standard pluripotency-promoting culture conditions containing Activin and collected at the beginning of the experiment. Mean ± SEM, n=3 cultures. Significant differences vs same iKD line in control conditions were calculated by 2-way ANOVA with post-hoc Holm-Sidak comparisons: **=p<0.01, and ***=p<0.001. (b) Western blots of cells treated as described in panel a. TUBA4A (α-tubulin): loading control. Results are representative of two independent experiments. (c) Measurement of mRNA stability in WTAP iKD hESCs cultured in absence (CTR) or presence of tetracycline (TET) for 10 days. Samples were collected following transcriptional inhibition using Actinomycin D (ActD) for the indicated time. The statistical significance of differences between the mRNA half-lives in TET vs CTR is reported (n=3 cultures, comparison of fits to one phase decay model by extra sum-of-squares F test). The difference was significant for NANOG but not SOX2 (95% confidence interval). (d) Model showing the interplays between Activin/Nodal signalling and m6A deposition in hPSCs (left), and the phenotype induced by impairment of the m6A methyltransferase complex (right).