Extended Data Figure 3: KBTBD8 controls neural crest specification.

a, Depletion of KBTBD8 from hESCs subjected to neural conversion results in loss of neural crest cells, as determined by immunofluorescence against HNK1, TFAP2 and p75 (n > 200 cells, mean of 3 biological replicates ± s.d.). b, H1 hESCs transduced with control (green) or KBTBD8 shRNAs (red) were subjected to neural conversion, and expression of neural crest markers SOX10 (circles) and SNAIL2 (boxes) was monitored by qRT–PCR (mean of 3 technical replicates ± s.e.m.). c, H1 hESCs described above were subjected to neural conversion, and abundance of CNS precursor markers SOX2 (circles) and PAX6 (boxes) was measured by qRT–PCR. d, H1 hESCs described above were subjected to neural conversion, and abundance of telencephalon markers SIX3 (circles) and FOXG1 (boxes) was measured by qRT–PCR. e, Expression of OCT4 was monitored by qRT–PCR during neural conversion in the presence or absence of KBTBD8. f, hESCs stably expressing control or KBTBD8 shRNAs were subjected to neural conversion and analysed for expression of pluripotency (OCT4, CDH1), neural crest (SOX10, SNAIL2, AP2), or CNS precursor markers (PAX6) by western blotting. To provide consistency, samples were taken from the same experiment as shown in Fig. 5d (asterisks mark blots that are also shown in Fig. 5d). g, Loss of neural crest occurs in response to KBTBD8 depletion by two independent shRNAs, as shown by western blot analysis. h, hESCs were subjected to neural conversion and analysed by immunofluorescence microscopy against SOX10 (neural crest), PAX6 (CNS precursor), and OCT4 (pluripotency) (confocal, original magnification 20×).