Fig 2: Tet1 maintains self-renewal by targeting Myc in iHepSCs. A, B The expression of Myc was reduced after the expression of Tet1 was knocked down by shRNA, as detected by qRT-PCR (A) and Western blotting (B). C The level of 5hmC in iHepSCs treated with the indicated dosage of TET-IN-C35 for 6 days was analyzed by dot blot. Each sample was loaded with 500 ng genomic DNA. D The expression level of Myc in iHepSCs treated with TET-IN-C35 was measured by qRT-PCR analyses. E The overexpression of Myc recovered the proliferation capacity of Tet1-KD iHepSCs. F The overexpression of Myc increased the colony numbers of Tet1-KD iHepSCs. G The overexpression of Myc increased the sphere numbers of Tet1-KD iHepSCs. The data are shown as the mean ± SEM, n = 3, Student’s t test (A, B, F and G) and Dunnett’s test (D), *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant

Fig 3: Tet1 maintains a high level of 5hmC in iHepSCs. A The results of qRT-PCR showed that the expression of Tet1 mRNA was repressed by shRNA. B The expression of Tet1 protein was determined by Western blot. C The quantification of the relative intensities of blots (B) showed that the protein expression of Tet1 was significantly downregulated. D The results of dot blot showed that the level of genomic 5mC was upregulated when the expression of Tet1 was downregulated. Each sample was loaded with 500 ng genomic DNA. E The results of dot blot showed that the level of genomic 5hmC was downregulated when the expression of Tet1 was inhibited. Each sample was loaded with 500 ng genomic DNA. The data are shown as the mean ± SEM, n = 3, Student’s t test, **P < 0.01, ***P < 0.001

Fig 4: Tet1 maintains the self-renewal of iHepSCs. A CCK 8 assay showed that the proliferation capacity of iHepSCs was inhibited after downregulating the expression of Tet1 with shRNA. B Representative images of colony formation of iHepSCs-scramble and Tet1-KD iHepSCs (left), and the numbers of colonies were significantly reduced in iHepSCs after downregulating the expression of Tet1 (right). C Representative plots (left) and statistical chart (right) of the percentage of G0/G1, S, and G2M cells of iHepSCs-scramble and Tet1-KD iHepSCs. The cell cycle of Tet1-KD iHepSCs exhibited obviously arrested G0/G1 compared to iHepSCs-scramble. D Representative plots (left) and statistical chart (right) of apoptosis of iHepSCs-scramble and Tet1-KD iHepSCs. E Representative images of spheres of iHepSCs-scramble and Tet1-KD iHepSCs (left), and the numbers of spheres were significantly reduced in iHepSCs after downregulating the expression of Tet1 (right). The data are shown as the mean ± SEM, n = 3, Student’s t test, *P < 0.05, **P < 0.01

Fig 5: Tet1 regulates the expression of Myc by directly binding to the CBS-1 and site A regions of the Myc promoter and demethylating the CpG cytosine. A Schematic diagram of the position of identified cis-elements that participate in the regulation of Myc expression on murine chromosome 15. Red rectangles represent CBS-1 and CBS-2. Blue arrows represent two initiating sites of transcription (P1, P2). Green rectangle indicates site A. The blue double-sided arrows represent the sites of primer sets used in Tet1-ChIP PCR. B ChIP-PCR revealed that TET1 was enriched in the CBS-1 and site A regions rather than the TATA box and CBS-2 regions in iHepSCs (n = 6, Student’s t test, ***P < 0.001). C Schematic diagram of methylated CpG cytosine in the sequence of CBS-1 and site A core region in MEFs, iHepSCs and Tet1 knockdown iHepSCs detected by BSP. The levels of methylation of cytosine in both regions of iHepSCs were lower than those of MEFs and Tet1-knockdown iHepSCs. The circles represent CpG dinucleotides. The filled circle is methylated cytosine, and the empty circle represents unmethylated cytosine. Ten clones of each sample were sequenced. Circles filled with dark blue ~ 100% positive; Circles filled with light blue 50–70% positive, empty circle ~ ≤ 30% positive. D The results of Pol II ChIP-PCR showed that the enrichment of Polymerase II at site A in iHepSCs was higher than that in MEFs. E The results of ChIP-PCR showed that the enrichment of H3K4me3 at site A in iHepSCs was higher than that in MEFs (n = 6, Student’s t test, **P < 0.01). F The results of ChIP-PCR showed that the enrichment of H3K27me3 in iHepSCs and MEFs had no difference (n = 6, Student’s t test)