Fig 2: Embryonic stem (ES) cell marker expression in KSCs.(A) Nanog, Oct4/Pou5f1, and Sox2 gene expression in KSCs and RTECs. Data represents the mean fold change ± standard error of the mean (SEM) when comparing KSCs to RTECs. All values were normalized to Gapdh expression. (**P < 0.01). B: Western blot analysis of ES markers in KSCs and RTECs. ß-actin was used as an internal control.

Fig 3: FOXO3 regulates the CSC phenotype through transcriptional inhibition of SOX2.A Western blot was performed to detect the expression of CD44, ALDH1A1, and BMI1 in FOXO3 overexpression sphere cells. B Western blot analysis of the expression of OCT4, NANOG, and SOX2 in FOXO3 overexpression sphere cells. C Real-time PCR was conducted to detect mRNA expression of SOX2 in FOXO3 overexpression sphere cells. D The binding motif of FOXO3. E The potential binding sites of FOXO3 in SOX2 promoter region. F Dual-luciferase reporter assay was performed to verify the binging sequence of FOXO3 in the SOX2 promoter region. G FOXO3 overexpression sphere cells were injected subcutaneously in the BALB/c nude mice. The tumor-initiating frequency was determined by ELDA software. n = 8. H Tumor volumes of negative control group and FOXO3 group (cell dose = 1 × 105). I The expression of FOXO3, SOX2, and CD44 in xenograft tumors was detected via immunohistochemistry. Scale bars, 200 µm. J The correction analysis between FOXO3 and SOX2 expression in xenograft tumors was determined via Spearman rank correlation analysis. n = 23. K The correction analysis between SOX2 and CD44 expression in xenograft tumors was determined via Spearman rank correlation analysis. n = 23. Data are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: ETV4 is required for breast cancer cell stem-like characteristics.A GSEA plot displaying that the ES1 target genes were strongly enriched in TCGA breast cancer tissues with high ETV4 expression. B Heatmap of the expression signature of PEA3 subfamily members between stem cell and non-stem cell subpopulations of MCF10A and SUM149 cells using gene expression data from GSE15192 and GSE132083. C Quantitative RT-PCR measurement of cancer stem cell markers (OCT4 and NANOG) and PEA3 subfamily members (ETV1, ETV4 and ETV5) in monolayer MDA-MB-231 cells and their spheres, monolayer MCF7 cells and their spheres. D Immunoblots of ETV4 and cancer stem cell markers (OCT4, NANOG and LIN28) in monolayer MCF7 cells and MCF7-derived spheres. E Quantitative RT-PCR measurement of ETV4, OCT4 and NANOG in MCF7 spheres and re-adherent MCF7 cells. F, G Representative images of spheres formed by the indicated cells with or without ETV4 depletion (F) and histograms showing the diameters and numbers of spheres (G). Scale bar =100 µm. H Representative images of spheres of MCF7 cells with or without ETV4 overexpression (left panel) and boxplots or histograms showing the diameters and numbers of spheres (right panel). A box represents the distribution of sphere diameter and a upper (lower) whisker represents the max (min) diameter. Scale bar =100 µm. I The subpopulation of CD44+/CD24- cells in control and ETV4-silenced MDA-MB-231 cells as assessed by flow cytometry. J Immunoblots of C-MYC, OCT4, NANOG and LIN28 in MDA-MB-231 and MCF7 cells with or without ETV4 knockdown. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: OCT4 IHC staining and survival analysis (A) Representative IHC staining of OCT4 in benign ovarian cyst tissues, OC tissues, and matched omental metastatic tissues. (B) Quantification of OCT4 H-score in different tissues. (C) OS of OC patients with low/high OCT4 H-score. (D) Expression rate of OCT4 in OC with or without lymph node metastasis. (E) Expression rate of OCT4 in high/low histological grade OC. (Data were expressed as the mean ± SD. Significance was calculated using Student’s t-test. ****, P<0.0001.)