Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901
Suppression of MEK/ERK signaling promotes self-renewal and colony morphology of mESCs. (a) PD promotes colony morphology of mESCs. J1 mESCs were treated with 1 μM PD or equal volume of DMSO for 24 h. Morphological changes were observed and recorded under a phase contrast microscope. Scale bar = 50 μm. (b) PD influences the expression pluripotent factors. ESCs were treated with or without 1 μM PD for 24 h; then the expression levels of Nanog, Tfcp2l1, Klf4, c-Myc, and Egr1 were analyzed by RT-qPCR. Gapdh was used as a normalization control. Error bars indicate mean ± SD of three independent experiments, compared with controls. (c) PD antagonizes RA-induced differentiation of ESCs. ESCs were treated with the indicated concentration of PD and/or together with 1 μM RA for 24 h; equal volume of DMSO was added for control samples. Then the protein expression levels of Nanog, Klf4, and c-Myc were analyzed by western blot. Gapdh was used as a normalization control. (d) PD do not influence epigenetic regulation of ESCs. ESCs were treated with the 1 μM PD or equal volume of DMSO for 24 h. Immunofluorescence staining assay was used for analysis of the expression level of 5hmC, Tet1, Ezh2, and H3K27me3. Nuclei were stained with DAPI; scale bar = 50 μm.
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