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. 2015 Jul 15;10(7):e0133444. doi: 10.1371/journal.pone.0133444

Fig 4. Functional analysis of H2B monoubiquitination in embryonic stem cell (ESC) differentiation.

Fig 4

a. Gene set enrichment analysis revealed that genes encoding proteins involved in cell differentiation were enriched following RNF20 knockdown and H2BK120R overexpression in HEK293T cells. b. Mouse embryonic stem cells (mESC) transfected with H2BWT or H2BK120R mutant plasmids were treated with or without LIF and RA, as indicated, to maintain ES status (LIF+) or to induce differentiation (RA+). Cells were lysed and analyzed by western blot with the specified antibodies. c. Microarray analysis of the mouse embryonic stem cells, as treated in b, was performed. d. Gene ontology (GO) and pathway analyses were performed using the MAS 3.0 online service. e. H2BK120R mutant ES cells showed impaired differentiation potential compared with the H2BWT ES cells. H2BWT or H2BK120R ES cells were treated with or without LIF and RA, as indicated, for 5 days. Cells were then subjected to alkaline phosphatase (AP) staining assay (upper panel). RNF20 knockdown inhibits ES cell differentiation. Control or RNF20-specific shRNA transfected ES cells were treated with or without LIF and RA, as indicated, for 5 days. The morphology of ES colonies was then analyzed by microscopy.