Figure 5.
STAT3 directly regulates Oct4 expression to maintain ESC pluripotency. (A,B) Immunodetection of pSTAT3 and OCT4 in 5-d in vitro blastocyst outgrowths of Stat3+/+ and Stat3mat−/−. Bar, 100 μm. (C) Nuclear expression of pSTAT3 and OCT4-GFP in ESCs. Bar, 20 μm. (D) Transfection of Stat3 shRNA results in down-regulation of Stat3 and Oct4 mRNA levels. (E) Stat3 knockdown in Oct4-Gfp ESCs results in decreased levels of STAT3, pSTAT3, and OCT4 by Western blot analysis. (F) Transfection of Stat3 shRNA into Oct4-Gfp ESCs leads to down-regulation of OCT4-GFP. Bar, 50 μm. (G) Reduced alkaline phosphatase detection in Stat3 knockdown Oct4-Gfp ESCs. Bar, 50 μm. (H) Treatment of iPSCs with STAT3 inhibitor STA-21 results in down-regulation of Oct4 mRNA. Treatment with actinomycin D or with actinomycin D plus a STAT3 inhibitor does not produce any additional effects. (I) STAT3 binds to the distal enhancer of Oct4 in mouse iPSCs. ChIP was performed on sonicated chromatin from iPSCs using pSTAT3 antibody. Immunoprecipitated DNA was analyzed by qRT-PCR with primer sets designed to detect ChIP-enriched DNA fragments (red boxes), shown within the context of the genomic structure of mouse Oct4. Amplicons are numbered in order, relative to their sites along the gene. Fold enrichment is the relative abundance of DNA fragments at the indicated regions over control region as quantified by real-time PCR. Standard deviations were calculated from biological replicates of qPCR data. (J) Luciferase results show that STAT3 could activate gene expression driven by the Oct4 promoter enhancer (P-E) in STAT3-overexpressing 293T cells in response to OSM. Oct4 P-E is the pGL3 luciferase vector containing the 4.6-kb upstream (P-E) region of Oct4. M67 and M67Δ are control luciferase vectors with the STAT3- and mutated STAT3-binding sites, respectively.