Fig 2: Influence of reduced neural stemness via SETDB1 knockdown on the property of HCT116 cells.A, phenotypic change in HCT116 cells after SETDB1 knockdown and cultured in normal and serum-free medium in a period as indicated. B and C, IB (B) and IF (C) detection of protein expression in control and knockdown cells. In (C), cell nuclei were counterstained with DAPI. D–F, tumor formation of control and knockdown cells in each six injected mice (D) and difference in tumor weight (E) and volume (F) between the two groups. Significance of difference in tumor weight (E) was calculated using unpaired Student’s t test. Significance of difference in tumor volume (F) was calculated using two-way ANOVA-Bonferroni/Dunn test. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001. G, IHC detection of cell/tissue markers in sections of tumors derived from control and knockdown cells. Objective magnification: 20 × . IF, immunofluorescence; IHC, immunohistochemical; IB, immunoblotting.

Fig 3: TRAF4 only interacts with the Tudor domain of SETDB1. (A) Schematic representation of full-length and deleted fragments of SETDB1. (B,C) 293FT cells infected with Flag-TRAF4 and MYC-SETDB1-WT/MYC-SETDB1-?SET/MYC-SETDB1-Tudor were immunoprecipitated with (B) anti-MYC antibody/(C) anti-Flag antibody, followed by immunoblotting. The interaction of TRAF4 with deleted fragments of SETDB1 (?SET and Tudor) were examined. (D) Schematic representation of deleted fragments of SETDB1. (E,F) 293FT cells infected with Flag-TRAF4 and MYC-SETDB1-Tudor/MYC-SETDB1-MBD/MYC-SETDB1-SET were immunoprecipitated with (E) anti-MYC antibody/(F) anti-Flag antibody, followed by immunoblotting. The interaction of TRAF4 with deleted fragments of SETDB1 (Tudor, MBD, and SET) were examined; + positive signal, * non-specific signal.

Fig 4: Influence of enhanced neural stemness via SETDB1 overexpression on the property of SW480 cells.A, comparison of phenotypic change between control (Vector) SW480 cells and cells with SETDB1 overexpression (SETDB1), which were cultured in normal and serum-free medium, respectively, in a period as indicated. B, protein expression change between control and overexpression cells, as detected with IB. C–E, tumor formation of control and overexpression cells in each six injected mice (C) and difference in tumor weight (D) and volume (E) between the two groups. Significance of difference in tumor weight (D) was calculated using unpaired Student’s t test. Significance of difference in tumor volume (E) was calculated using two-way ANOVA-Bonferroni/Dunn test. Data are shown as mean ± SD. *p < 0.05, ***p < 0.001. F–H, difference in transcription of genes representing neural stemness (F), neuronal differentiation (G), and mesodermal and endodermal tissue differentiation (H) between tumors of control and of overexpression cells, as detected with RT-qPCR. Significance in transcription change was calculated based on experiments in triplicate using unpaired Student’s t test. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant. I, IHC detection of cell/tissue markers in sections of tumors derived from control and overexpression cells. Objective magnification: 20 × . IB, immunoblotting; IHC, immunohistochemical; RT-qPCR, Reverse transcriptase-quantitative PCR.

Fig 5: Characterization of xenograft tumors formed by SW480 cells using serial transplantation and a general model unifying embryogenesis and tumorigenesis.A–C, tumor formation via serial transplantation in each six injected mice (A) and difference in tumor volume (B) and weight (C) between different groups. Significance of difference in tumor volume (B) was calculated using two-way ANOVA-Bonferroni/Dunn test. Significance of difference in tumor weight (C) was calculated using unpaired Student’s t test. Data are shown as mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. D, comparison of expression of genes for SETDB1 and neural stemness protein in SW480 cells and xenograft tumors in (A), as revealed by RT-qPCR. Significance in transcription change was calculated based on experiments in triplicate using unpaired Student’s t test. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS, not significant. E, IHC analysis of SETDB1 and other marker protein expression in sections of tumors in (A). Objective magnification: 20 × . F, a model depicting the correlation between embryonic development and tumorigenesis. Normal embryogenesis needs the inhibition of epidermal fate in gastrula ectoderm by extracellular signals secreted by Spemann organizer or node, leading to restoration of the neural ground state or neural stemness in ectoderm, a process called neural induction. Formation of the neural precursor tissues is required for differentiation of the nervous system and many nonneural tissues, such that the body axis of an embryo can form. This neural induction can occur ectopically during embryogenesis, caused by either an ectopic organizer or node activity or ectopic expression of embryonic neural genes, leading to formation of secondary embryonic structures or a conjoined twin. This process might occur in any cell and at any time of an animal life. Somatic cells could suffer various extracellular (e.g., microenvironmental change) and/or intracellular (e.g., gene mutations) insults. If occasionally the insults cause activation of neural stemness regulatory network and/or downregulation/silencing of tissue-specific or differentiation genes/factors, then somatic cells progressively lose their original cell identity and restore the neural ground state or neural stemness, similar to the neural induction process in gastrula ectodermal cells. The resulting cells can self-renew and differentiate, resembling a defected process of embryonic development, that is, tumorigenesis. For detailed information, see text and also refer to refs. 1 and 13 (1, 13) and references therein. IHC, immunohistochemical; RT-qPCR, Reverse transcriptase-quantitative PCR.