Fig 2: Neuronal differentiation of primNSCs induced by RA treatment and by knockdown of Setdb1.A and B, neuronal differentiation phenotype in ESC-derived primNSCs that were treated with RA at an indicated dose and cultured in NSC-specific serum-free medium for a period as indicated (A), and IB detection of protein expression in control (DMSO) and treated (RA) cells (B). C and D, neuronal differentiation phenotype in ESC-derived primNSCs induced by knockdown of Setdb1 and cultured in serum-free medium (C), and IF detection of protein expression in control (shCtrl) and knockdown (shSetdb1) cells (D). E–G, neuronal differentiation phenotype in NE-4C cells induced by knockdown of Setdb1 and cultured in serum-free medium (E), and IF (F) and IB (G) detection of protein expression in control (shCtrl) and knockdown (shSetdb1) cells. H and I, enrichment analysis on GO and KEGG pathway terms for upregulated (H) and downregulated (I) genes identified in a transcriptome profiling on control and knockdown cells in (E). In (D) and (F), cell nuclei were counterstained with DAPI. In (F), Map2 and Tubb3 were simultaneously detected with cells on the same coverslips, either control (shCtrl) or knockdown cells (shSetdb1). ESCs, embryonic stem cells; IF, immunofluorescence; IB, immunoblotting; NSCs, neural stem cells; primNSCs, primitive NSCs; RA, retinoic acid.

Fig 3: 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 4: Selection of targeted genes by C11orf46-SETDB1 complex. a Mouse neuroblastoma cells NSC34 inducibly expressing C11orf46 shRNA upon doxycycline addition (schematic in left panel); selected subset of 17 differentially expressed genes implicated in axonal growth and development was tested in RNA expressed from NSC34:Tet-On C11orf46 shRNA cell (right panel). b Neuronal progenitors received control or C11orf46 shRNA and GFP marker plasmids at somatosensory cortex by IUE at E15; four days later at P0 GFP-positive cells were collected by fluorescence-activated cell sorting (FACS). c C11orf46 transcript levels were reduced by 50% in sorted GFP-positive cortical neurons electroporated with C11orf46 shRNA1 as compared to GFP-positive neurons with control shRNA (P = 0.0003). Note increase of Sema6a transcript upon C11orf46 knockdown (P = 0.0085). *P < 0.05, **P < 0.01, ***P < 0.001 determined by Student’s t-test test. n = 10–19 independent mice per condition. All data were normalized by the Hprt transcript. Bar graphs indicate mean ± S.E.M (a, c)

Fig 5: 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.