Additional file 1 of AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts
Creators: name: Francia, Marcos Gabriel
name: Verneri, Paula
name: Oses, Camila
name: Vazquez Echegaray, Camila
name: Garcia, Mora Reneé
name: Toro, Ayelen
name: Levi, Valeria
name: Guberman, Alejandra Sonia
Publisher: name: figshare
Year: 2024
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Developmental Biology
subject: Cancer
subject: Science Policy
subject: Mental Health
subject: Computational Biology

description: Additional file 1: Figure S1. Nanog expression and promoter state in MEF and PSCs. Data analysis of Nanog expression and epigenetic marks on its promoter in ESCs, iPSCs and MEFs. Stemformatics data-mining platform was used to perform the analysis from publicly available data [1, 2]. Nanog expression levels from RNA-seq (transcript) and LC-MS (protein) are shown in the upper panel; epigenetic marks within the Nanog promoter region from Histone ChIP-seq (H3K4me3 and H3K27me3, associated to active promoters and repressive marks, respectively) and from cytosine methylation by Bisulfite-sequencing are shown in the lower panel. Figure S2. Summary of SUMOylatability and previous results of the AKT variants used. Schematic representations of the AKT1 variants used in our current and previous work and table summarizing their main features (SUMOylation capability [3] and effect on the Nanog promoter in ESCs [4]). SUMO is represented by yellow balloons, hyperactivity-inducing mutations are portrayed as spiny edges and the two lysines replaced by arginine in 2KR and E17K/2KR mutants are indicated by the red Xs. Figure S3. NANOG is not detected in the U-2 OS cell line. Representative epifluorescence microscopy images of NANOG immunofluorescence (IF) of the tumoral osteosarcoma U-2 OS cell line, transfected with a vector encoding eGFP-NANOG. Transfection and IF were performed as previously described [4]. Scale bar represents 10 μm. The images show that endogenous NANOG is not detected, since the non-transfected cell (left, yellow arrow), evidenced by DAPI staining, has no NANOG signal (right panel). As a control of the IF, cells were transfected with a vector encoding NANOG fused to the fluorescent protein eGFP (eGFP-NANOG). This fusion protein was detected both through detection of eGFP fluorescence (middle panel) and by IF against NANOG (right panel). Figure S4. OCT4 and SOX2 are not detected in MEFs and U-2 OS cells. Western blot analysis of protein extracts of ESCs, NIH/3T3 MEFs and U-2 OS evaluating the presence of OCT4 and SOX2. GAPDH was revealed as loading control. The sample loaded in the 3rd lane corresponds to a cell line that is not included in this work. Figure S5. Downregulation of Oct4, Sox2 and Nanog by shRNA. ESCs were transfected with the pLKO.1-puro derived vectors encoding shRNA targeting Oct4 (shOct4), Sox2 (shSox2), Nanog (shNanog) and eGFP (shGFP), as indicated below each bar. mRNA levels of each specific target, indicated at the top, were evaluated by RT-qPCR, normalized to the geometric mean of Gapdh and Pgk1 and referred to the control (shGFP). Bars represent the mean ± SEM of three independent experiments. Asterisks (*) indicate significant differences compared to the corresponding control condition (p &lt; 0.05). Figure S6. Multi-omic analysis of genes from Groups III to V in ESCs differentiating to epiblast-like cells (Related to Figure 3). mRNA (green) and/or protein (light blue) expression levels (log2 fold change) from multi-omic analysis of ESCs differentiating to epiblast-like cells. Data analysis was performed in the Stem Cell Atlas data-mining platform ( http://www.stemcellatlas.org/ ) from publicly available transcriptomic and proteomic data [5]. Table S1. Full gene list of Group I from the cross-matching analysis of Figure 3.