Fig. 2

Downregulation of the pluripotency TFs Oct4, Sox2 or Nanog does not interfere with the SUMOylation-dependent AKT induction of Nanog promoter activity. A Oct4 and Sox2 gene expression analysis from an RNA-seq experiment in ESCs, iPSCs, and MEFs seq [45]. Data analysis was performed in Stemformatics data-mining platform from publicly available data. DT: detection threshold. B Cropped images of western blot analysis of protein extracts of NIH/3T3 MEFs and ESCs, evaluating the presence of OCT4 and SOX2. GAPDH was revealed as loading control (Full blots are shown in Additional file 1: Figure S4). C Visualization of the representative enrichment profiles (reads per million) of the indicated TFs in the 2.5 kbp region of the Nanog genomic locus included in Nanog5P reporter. These results correspond to the analysis of publicly available ChIP-seq data from experiments performed in ESCs retrieved from the Chip Atlas database [48,49,50]. Data was visualized using the Integrative Genomics Viewer (IGV) software. D Flowchart representing the rationale of the study to evaluate possible mediators of the SUMOylation-dependent AKT induction of Nanog reporter. Specifically, we evaluated the involvement of the pluripotency TFs through shRNA-mediated knockdown. Panel i shows the control case in which no factor is interfered, thus resulting in the induction of Nanog promoter by wt AKT due to the presence of the active mediator within the cell context. Panel ii shows the case when performing an inhibition of a specific factor not involved in this regulation, obtaining a similar response to the control condition. Panel iii shows that in the case of interfering with a key mediator, no effect on Nanog promoter by wt AKT is expected. Induction of the reporter by 2KR AKT1 is not expected in any of these cases. E Schematic diagram of the experimental design. F ESCs transfected with either an AKT1 variant or the empty vector (basal), along with the Nanog5P, and the pLKO.1-puro derived vectors targeting either eGFP, Oct4, Sox2 or Nanog, as indicated. The results were referred to the control condition (basal) and are shown as mean ± SEM of three independent experiments. Different letters indicate significant differences (p < 0.01)