Fig 2: Knockdown of Snail restrained the expression of EMT, anti-apoptosis and CSC propertiesA. Knockdown of Snail altered the expression of apoptotic-related proteins and reverses the expression of RKIP. B. Knockdown of Snail decreased the expression of MDR1 and ABCG2. C. Knockdown of Snail increased the expression of E-cadherin, decreased the expression of vimentin, indicating a reversal of EMT phenomenon. D. Knockdown of Snail suppressed the expression of CSC properties with decreasing the expression of Nanog, OCT4. E. Knockdown of Snail declined the ability of sphere formation.

Fig 3: LT-NNK exposure enriched CSC properties with presentation of CSC-representative markers and sphere formationA. Overexpression of CSC-representative markers including Nanog and OCT4 was found in NNK-treated cells in a dose dependent manner, compared with control cells by Western blotting. B. LT-NNK exposure also demonstrates increased expression of CSC-representative markers including CD133, CD44 and CD24 by flow cytometry. C. Using a nonadhesive culture system, LT-NNK exposure is prone to form sphere, compared with the control cells.

Fig 4: DR cells exhibiting CSC propertiesA. Using the nonadhesive sphere culture system, the derivative DR-H23 cells were cultured further for 14 days and generated a spheroid phenotype termed DR spheres (DRSPs). B. MTT assay showed that more surviving cells were significantly found and maintained in DRSPs than those of control cells (p < 0.05). Western blotting showed that the drug resistance-related genes ABCG-2 and MDR-1 were obviously upregulated in DRSPs compared with DR-H23 and control cells. C. Immunofluorescence assay and western blotting showed overexpression of CSC-representative markers, including CD44high/CD24low, CD133, Oct4, and Nanog, in DRSPs compared with DR-H23 and control cells. D. Flow cytometric analysis of ALDH1 showed significantly increased activity in DRSPs compared with DR-H23 and control cells (p < 0.05).

Fig 5: Generation and characterization of iPSCs derived from a patient with Best disease and an unaffected sibling. (A) Phase-contrast photomicrograph and alkaline phosphatase activity of undifferentiated control iPSCs (ctrl-iPSCs) and Best disease iPSCs (BD-iPSCs). Scale bar = 200 µm. (B) The RT-PCR results indicated an embryonic stem cell (ESC)-like gene expression pattern in representative colonies of iPSCs. Dental pulp-derived stromal cells (DR-SC) were used as a negative control, and human ES-H9 cells as a positive control. (C) Immunofluorescence staining demonstrated the expression of pluripotency markers (OCT4, TRA-1-61, Nanog, and TRA-1-81) in undifferentiated iPSCs from patient and unaffected sibling. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. (D) Immunofluorescence staining demonstrated markers of all three germ layers that were expressed in spontaneously differentiated iPSCs. Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. (E) Sequence analysis revealed the existence of specific Best mutations in the BD-iPSC lines.