Fig. 1: ID1 and ID3 double knockout impairs the survival and promotes ectoderm specification of primed hESCs.

A Clone morphology of the WT and double knockout lines after culturing for 4 days and the clone outgrowth of the WT and double KO lines after supplementation with or without Y27632, bar = 300 μm. B Number of the WT and IDs KO cells after four days of culture with or without Y27632 (n = 3), **p < 0.01 compared with WT, t-test. C Pluripotent markers expression level of the WT and double knockout lines after spontaneous differentiation at day 0 and day 3 (n = 3), *p < 0.05; **p < 0.01 compared with day 0 WT; #p < 0.05; ##p < 0.01 compared with day 3 WT, one-way ANOVA followed by t-test. D mRNA levels of mesoderm, endoderm, and neuroectoderm markers in the WT and double knockout lines were measured via qPCR at the indicated time points, as the cells spontaneously differentiated into embryoid bodies (EBs); the results were normalized to measurements for the WT cells at the beginning of the differentiation period (n = 3). *p < 0.05; **p < 0.01 compared with the WT; one-way ANOVA followed by t-test. E Immunofluorescence analysis of PAX6 expression levels in the WT and KO lines after spontaneous differentiation, bar = 300 μm. F The protien levels of NANOG and PAX6 were detected by immunoblotting after three days spontaneous differentiation. G Immunofluorescence analysis of NESTIN and TUJ1 (β3-TUBLIN) expression levels in the WT and double KO lines after spontaneous differentiation, bar = 200 μm. H Detection of calcium influx in differentiated neurons after stimulation with glutamic acid.