Fig 2: SOX2 and FOXO3 co-regulate a stemness network in neural progenitor cells (NPCs) and GBMs. (A) Landscape in silico deletion analysis analysis of the FOXO3-associated The Cancer Genome Atlas Project (TCGA) GBM network identifies candidate transcriptional co-regulators. The top 10 most significant transcription factors are shown. The p-value of the most enriched dataset for each factor is represented. (B) Venn diagram depicting significant overlap between the genes that are direct targets of SOX2 and FOXO3 in NPCs. p = 4.24 × 10-125, Fisher’s exact test. (C) PANTHER biological process analysis of the 2064 shared direct targets of FOXO3 and SOX2. GO terms displayed based on the hierarchical organization with Bonferonni corrected p < 0.05. (D) Enrichment of the shared NPC network, FOXO3-specific network, SOX2-specific network, or non-targets in the neuronal stem cell population maintenance term (GO:0097150). (E) GSEA analysis reveals enrichment for genes involved in NSC quiescence or activation are upregulated in tumors with high FOXO3/SOX2 expression. (F) Stemness mRNA signature index in FOXO3/SOX2 high and low tumor samples. Mann–Whitney test at p < 0.05, *p = 0.0271. (G) GSEA plot shows an enrichment for quiescence signatures and depletion of activated signatures in samples with high expression of FOXO3/SOX2 regulated by the FOXO3-specific network. (H) Survival analysis of FOXO3/SOX2 high TCGA GBMs, compared to all other tumors in the first 500 days. **p = 0.0024. Log-rank (Mantel–Cox) test performed on all survival analyses with p < 0.05 considered significant. Censored points shown are days until the last follow-up where days until death was not applicable/available

Fig 3: Evaluation of cellular identity and morphology in 3D brain organoid.A Representative immunostaining showing the presence of a N-CADHERIN-positive (green) germinal layer in correspondence of the apical membrane of the ventricular zone layer in a FMRP-WT brain organoid at day 50. B, C Representative images revealing forebrain progenitor markers in developing cortical plates within FMRP-WT brain organoid at day 50: B SOX2 (green), DAPI (blue); C PAX6 (red), FOXG1 (green) and DAPI (blue). D Representative staining of early-born deep-layer cortical neurons positive for TBR1 (red), CTIP2 (green) markers, and pan-neuronal MAP2 marker (white) in a FMRP-WT brain organoid at day 50. E Representative images of a cortical plate from a FMRP-WT brain organoid at day 100 revealing heterogeneous cellular population, labeled for specific cortical neuronal marker (CTIP2, green), astrocytic marker (GFAP, red), and neuronal progenitor marker (PAX6, white). Nuclei were stained with DAPI (blue). Scale bar: 100 µm.

Fig 4: Representative images of post-warmed expanded and hatched blastocysts derived from cow-derived D7 expanded blastocysts vitrified/warmed with vitrification media supplemented with 100 µg/mL EPS ID1. DAPI (blue), SOX2 (red), and TUNEL (green) staining were examined using DAPI, SOX2-Alexa Fluor, and FITC filters, respectively, for total (A1,A2), ICM (B1,B2), and apoptotic (C1,C2) cell counts. An overlay is provided in (D1,D2). (A1,B1,C1,D1): Expanded blastocyst; (A2,B2,C2,D2): Hatched blastocyst. Scale bar = 30 µm. DAPI (406-diamidino-2-phenylindole), TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling).

Fig 5: Effect of survivin inhibition on stemness. (A) The expression of stemness marker Sox2 in PIP treated cells using RT-PCR (B) Clonogenic potential in the isolated GSCs with single drug and combination treatments. (C) Representative images of colony formation in the S pool with and without treatment. The graphical data are presented as mean ± SD and the statistical analysis was performed using one-way ANOVA (***: p-value < 0.001). The scale bar indicates 20 µm.