iPSC colony numbers are from three biological replicates. ∗∗p<0.01 compared with mutant OCT4 #p<0.01, #p<0.001compared with hnRNPA- or FUS-IDR. Mutations of OCT4 reduce the reprogramming efficiency, which is restored by IDR fusion. (D) CFA is performed to compare the influence of WT, mutant and IDR-fused OCT4 on reprogramming efficiency. 20uM proteins were added into droplet formation buffer with 125 mM NaCl and 10% PEG8000. (A–C) Representative images of droplet formation showing that OCT4 can form droplets in vitro with MED1-IDR. Phase separation regulates cell fate transitions These results support that our approach can effectively control phase-separated capacity of proteins. Furthermore, the disabled phase separation ability of OCT4 mutants can be rescued by fusing an IDR of FUS or hnRNPA ( Wang et al., 2021). Both acidic mutation and residue deletion can disrupt OCT4 condensates in vivo and OCT4 droplets in vitro, demonstrating IDR perturbations can destroy the phase separation capacity of proteins. These claims are also validated by in vivo study showing that OCT4 can form phase-separated condensates in vivo, which can fast recover after photobleaching ( Wang et al., 2021). ![]() ![]() OCT4 droplets can fuse, and can fast recover after photobleaching, indicating OCT4 droplets have liquid-like behavior. These results demonstrate that OCT4 phase separation needs the participation of coactivator such as MED1. The IDR of MED1 is required for OCT4 to form circle-like droplets ( Figures 4B and 4C). OCT4 alone can form irregular aggregates ( Figure 4A), which is not associated with phase separation. GelCode TM Blue Safe Protein Stain Buffer Chemicals, peptides, and recombinant proteins
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