Fig 2: Tnfaip2 deletion abrogates differentiation potential of ESCs A–FOne thousand ESCs of the indicated genotypes were placed into hanging drop cultures to form embryoid bodies (EBs). After 3 days (referred to as day 0), differentiation was induced by exposure to differentiation medium (days 1 to days 3 after medium change). (A) Representative photographs of EBs of the indicated genotypes at day 0 and day 3 after differentiation induction (experiments were conducted in three biological replicates; n = 5–42 EBs per biological replicate, scale bar: 0.02 mm). (B–D) RNA-seq analysis was conducted on Tnfaip2 -/- EBs versus WT EBs on day 3 of differentiation induction of EBs (n = 3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets retrieved from the AmiGO 2 data base (Carbon et al, 2009): (B) “stem cell maintenance: positive and negative regulators”, (C) “mesoderm development” and (D) “ectoderm development” from the AmiGO 2 data base (Carbon et al, 2009). Tnfaip2 and 5 other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42, and Klf2) were additionally incorporated based on literature searches. The color scale represents the gene-wise z-score calculated from normalized gene expression levels. The asterisk refers to the gene identified in this study. (E) Representative image of immunofluorescence staining against the ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) in WT and Tnfaip2 -/- EBs on day 3 of differentiation induction (3 repeat experiments were conducted on a total number of 9–10 EBs per genotype, scale bar: 20µm). (F) mRNA expression of Tnfaip2 measured by RT–qPCR in ESCs on the indicated days of differentiation induction of WT EBs [n = 3 biological replicates; log-transformed data were normally distributed (P > 0.05 as per Shapiro–Wilk test), the P-value for the upregulation of Tnfaip2 on consecutive time points (days 0, 2, and 3) was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, P 1,b = 0.1 (bidirectional) and P 1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some time point is P triple,tp = P 1,b · P 1,u · P 1,u, and finding such a triple somewhere across the 4-step time series is P triple = P triple,tp + (1-P 1,u) · P triple,tp = 0.0005.

Fig 3: SOX1 is not regulating the WNT signaling pathway in glioblastoma.(A) Representative images of CTNNB1 (ß-catenin) immunofluorescence staining in U251 plko and sh1 cells (n = 4). (B) Representative images of CTNNB1 immunohistochemical staining in U251 pLKO, sh1 and sh5 derived tumors (n = 4). (C) mRNA levels of CTNNB1, CCND1 (CYCLIN D1) and MYC in GNS166 pLKO and sh1 cells. qRT-PCR data are normalized to GAPDH expression (n = 2). (D) Scatter plot of log2 of the FPKM of CTNNB1, MYC and CCND1 vs SOX1 expression. In the x-axis, the correlation and its statistical significance are included. Only CCND1 has a significant correlation with SOX1.

Fig 4: SOX1 overexpression contributes to the malignant phenotype in GSCs.(A) SOX1 and SOX2 protein expression in GNS166 cells transduced with ectopic SOX1 (n = 3). (B) mRNA levels of the indicated stem cell markers in GNS166 cells transduced with SOX1 relative to control (GFP) expression (n = 3). (C) mRNA levels of the indicated differentiation markers in the same cells (n = 3). (D) Cell growth at day 5 comparing control (GFP) and SOX1 overexpressing GNS166 cells (n = 6). (E) Representative image and quantification of the number of p-H3 positive cells in SOX1 transduced GNS166 cells compared to control (GFP) condition (n = 6).

Fig 5: CMTR1 protein expression increases during ESC differentiation Differentiation was induced in ESC by LIF withdrawal. (A) Phase contrast images (top) and RT-PCR analysis of pluripotency-associated (Oct4, Nanog, Sox2, Klf4) and differentiation-associated (Brac, Sox17, Sox1) genes presented (bottom). (B) Representative western blot analysis of CMTR1 and pluripotency associated proteins (top) and quantification of CMTR1 RNA and protein levels (bottom). Neural differentiation was induced in ESC. (C) Phase contrast images (top) and RT-PCR analysis of pluripotency-associated (Oct4, Nanog, Sox2, Klf4) and neural (Pax6) genes presented (bottom). (D) Representative western blot analysis of CMTR1 and pluripotency associated proteins (top) and quantification of CMTR1 RNA and protein levels (bottom). For charts, average and standard deviation of 3 + experiments presented. For CMTR1 protein chart, ‘*’ indicates Student's T test P-value < 0.05. RNA levels were normalized to Actin and Gapdh and relative to day 0. Proteins levels were quantified relative to day 0 and normalized to Actin by densitometry using ImageJ.