Perturbation experiment of M-DNB factors in DE differentiation system
(A) Experimental flow of M-DNB factors’ perturbation and hESCs’ differentiation. Reference is the process of hESCs’ differentiation, and we obtained ME cells at 24 h, DE cells at 72 h of hESCs’ differentiation. Perturbation of M-DNB factors’ expression consists of two parts. In tipping point 1, we knock down FOS and overexpress HSF1 and MYCN, respectively, at 12 h (nearly) and obtained ES-FOS-KO cells, ES-HSF1-OE cells, and ES-MYCN-OE cells at 72 h of differentiation. In tipping point 2, we overexpress MYCN, respectively, at 36 h (nearly) and obtained ME-MYCN-OE cells at 72 h of differentiation.
(B) RNA expression levels of ES and ME stage marker genes.
(C) RNA expression levels of ME and DE stage marker genes. OCT4, and SOX2 as ES marker genes; EOMES, MIXL1, and GSC as ME markers; SOX17 as DE marker genes.