Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901
Transcripts involved in self-renewal and differentiation were regulated by PD. (a) qPCR validation of the microarray data. Cells were treated with 1 μM PD or equal volume of DMSO for 24 h. The expression levels of Prdm14, pramle7, Gata6, Cdx2, Wnt8a, and Dusp4 were detected by RT-qPCR. Error bars indicate mean ± SD of three independent experiments, compared with controls. (b) The expression of Oct4 and Sox2. Cells were treated with 1 μM PD or equal volume of DMSO for 24 h. Immunofluorescence staining assay was used for analysis of the expression level of Oct4 and Sox2. Nuclei were stained with DAPI; scale bar = 50 μm. (c and d) GO annotation of PD-regulated genes. GO term enrichment of the “biological process” category of PD-regulated genes. GO terms ranked according to the −Log2P of upregulated genes (count > 10) (c) or downregulated genes (d) were plotted. (e) KEGG pathway analysis of differentially expressed genes. KEGG pathway analysis of differentially expressed genes in PD-treated J1 mESCs. This result was ranked according to the −Log2P of PD-regulated genes (count > 10). (f) Dual-luciferase reporter assay to identify signaling transduction pathways regulated by PD. Pathway reporter vectors (including negative control) and internal control pRL-SV40 were cotransfected by Lipofectamine 2000. 24 h after transfection, 1 μM PD or an equal volume of DMSO was added to cell medium for another 24 h. Luciferase activity is presented relative to negative control pTA-luc. Data are presented as mean ± SD of three independent experiments, .
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