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Figure 1. Regulation of Nanog by Oct4. A) ES cells with overexpressed Oct4 or down-regulated Nanog share same morphology. R1 ES cells were transfected with U6-GFP, siNanog-GFP, or Oct4F plus U6GFP as indicated. Morphology of obtained clones was photographed in bright fields (left panels) or in fluorescence (right panels) as presented. B) Overexpression of Oct4 and down-regulation of Nanog induced identical differentiation pattern. ES cells transfected with GFP vector (lane 2), Oct4 (lane 3), Nanog siRNA vector (lane 4), and day 6 embryonic bodies (EB6, lane 5) were analyzed by reverse transcriptase-polymerase chain reaction for the expression of pluripotent as well as differentiation markers as indicated. Note identical pattern of marker expression between Oct4 and Nanog siRNA transfected cells. C) Nanog promoter (
1 kb) recapitulates the endogenous expression of Nanog. Endogenous Nanog transcripts in NIH3T3, P19, F9, and ES cells were analyzed by reverse transcription (RT)-polymerase chain reaction (PCR). Activity of Nanog promoter was also analyzed in these cell lines to show its specificity in pluripotent cells as indicated as described (16). D) Oct4 suppresses activity of Nanog promoter in F9 cells. Nanog promoter construct (NanP, lanes 2–5) and Oct4 reporter (6w-lu, lanes 6–9) were cotransfected with Oct4 expression constructs with increasing doses into F9 EC cells, and luciferase activities were measured and analyzed as described previously (16). E) Binding of Nanog promoter by Oct4 in ES cells. Chromatin immuno-precipitation analysis was performed to demonstrate in vivo binding of Nanog promoter by Oct4. F) Oct4 suppresses Nanog promoter constructs in NIH3T3 cells. Cotransfecton of Oct4 with reporters carrying deletions in the Nanog promoter was performed and analyzed as described in D. G) A mutation in the Oct4 binding site abolished regulation of Nanog promoter by Oct4. Reporters carrying wild-type and mutant promoters were cotransfected with Oct4 or Oct4 siRNA vectors and analyzed as in D. H) Overexpressed Oct4 reduces the expression of Nanog in ES cells. ES or Oct4 transfected ES cells were lysed and fractioned by 10% SDS-PAGE. Endogenous Nanog or transfected Oct4 protein was detected by anti-Nanog or anti-FLAG (Oct4 is FLAG tagged) antibodies, respectively. Levels of actin in the cell lysate were detected by antiactin antibody as a loading control. I) Regulation of Nanog by Oct4 siRNA in F9 cells. Nanog promoter construct (NanP, lane2–6) was cotransfected with increasing doses of Oct4 siRNA construct (lane 3–6) into F9 cells, and activities were analyzed by luciferase assay. J) Oct4 binding site is required for activity of Nanog promoter in F9 cells. Wide-type and mutant Nanog promoter constructs (lanes 2 and 3) were transfected to F9 cells, and their activities were evaluated by luciferase assay as in D.
