Fig 2: Analysis of cell growth and sphere formation in M1-2 and NHRI-HN1 cells. (A) Representative growth curves for M1-2 and NHRI-HN1 cells analyzed by MTS assay. The results were expressed as the fold change to the results at day 1. (B) Cell cycle analysis of M1-2 and NHRI-HN1 cells by Fluorescence Activated Cell Sorting (FACS). (C) Sphere-forming activity of M1-2 and NHRI-HN1 cells was assessed by sphere culture. Left: The presentative pictures for sphere formation at 50× magnification. Right: The average sphere diameter and number of spheres are shown. (D) Analysis of CSCs in M1-2 and NHRI-HN1 cells by FACS using anti-Prominin-1 and anti-CD44 antibodies. Representative data are shown in the left panel. The relative level of Prominin-1+/CD44+ cells is shown in the right panel. The Prominin-1+/CD44+ ratio was calculated by normalizing the mean percentage of Prominin-1+/CD44+ NHRI-HN1 cells to that of M1-2 cells. (E) Immunoblot analysis of stemness-related transcription factors Klf4, Nanog, and Oct4. The protein levels were normalized to internal control (a-tubulin). The ratios were calculated by dividing the normalized protein levels of NHRI-HN1 cells by those of M1-2 cells. Error bars represent SE; * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 3: Stemness markers expression in prostate cancer cells. SOX2, OCT4, NANOG, and CD133 gene expression were evaluated by real-time PCR in DU145 (A) and PC3 (B) cells. Data were normalized to GAPDH expression. Means ± SD (error bars), n=6. **p < 0.001, *p<0.05 vs. (C) SOX2, OCT4, and NANOG protein level were quantified by WB in DU145 and PC3 cells. GAPDH was included as loading control.

Fig 4: Ethidium bromide-stained agarose gel electrophoresis of RT/PCR products showing clearly defined bands corresponding to NANOG, OCT4, and SOX2 amplicons of the expected size (RPS18: ribosomal protein S18).

Fig 5: Derivation of cancer stem cells (CSCs) from OSCC cell lines via a sphere-forming assay and the characterization of their stem cell-like features. (a) The morphology of the parental SAS and H103 and their derived tumour spheres. SAS and H103 in normal culture media were observed as polygonal squamous epithelial cells with the adherent growth pattern. Within 7 d, tumour spheres, comprised of aggregated and suspended cells derived from SAS and H103, were formed in the specialized serum-free medium containing serum substitute, heparin, and growth factors and in a low attachment plate (100× magnification). The average diameters of the SAS and H103 tumour spheres were 133.4 ± 34.36 µm and 68.1 ± 13.37 µm, respectively. (b) Assessment of cell viability of SAS, SAS tumour spheres, and H103 after 72 h exposure to cisplatin. IC50 was defined as the concentration of cisplatin required to reduce cell viability by half. Higher IC50 values indicated lower sensitivity of the cells towards cisplatin and possibly cisplatin resistance. (c) Western blots of Sox2, Oct4 and ß-actin and the relative expression levels of the Sox2 and Oct4 transcription factors normalized to the ß-actin protein in SAS and SAS tumour spheres. The full-length blots are presented in Supplementary Figure S2. (d) Expression of CD338, CD117 and CD44 surface markers in both SAS and SAS tumour spheres, as analyzed by flow cytometry. Multi-staining flow cytometry was used to analyse the surface expression of CD338 and CD117 for (I) SAS and (II) SAS tumour spheres. Single-staining flow cytometry was used to analyse the surface expression of CD44 for (III) SAS and (IV) SAS tumour spheres. All the data are presented as mean ± SD. **P < 0.01, n = 3.