Fig. 6

MenSCs restored BNIP3/BNIP3L expression via TET2-mediate active demethylation. A The 5-mc levels of BNIP3 and BNIP3L promoter regions in HCC cells were determined using qRT-PCR followed with methylated DNA immunoprecipitation (MeDIP). MenSCs significantly reduced the methylation levels of the BNIP3 and BNIP3L promoters. Cells were cocultured with MenSCs under hypoxia for 48 h. B The mRNA levels of DNMT1, DNMT3A, DNMT3B, TET1, TET2, and TET3 in HCC-SR cells were determined using qRT-PCR analysis after 24, 48, and 72 h of coculture with MenSCs under hypoxia. C The levels of DNMT1, TET1, and TET2 in HCC-SR cells were determined using immunoblotting analysis after 48 h of coculture with MenSCs under hypoxia. According to B and C, the changes in TET2 transcription levels and protein levels appeared to be the most conserved between HCC-SR cell lines after MenSC treatments, which was stably upregulated by MenSCs in all HCC-SR cell lines. D TET2 activity in HCC-SR cells was determined using a TET Hydroxylase Activity Quantification Kit. MenSCs increased TET2 activity in HepG2-SR and Huh7-SR cells. Cells were cocultured with MenSCs under hypoxia for 72 h and 1ug of TET2 protein was purified using co-IP. E The levels of TET2, BNIP3, BNIP3L, and β-actin in Huh7-SR cells were determined using immunoblotting analysis. F The mRNA levels of BNIP3 and BNIP3L were determined using qRT-PCR analysis. G The 5-mc levels of BNIP3 and BNIP3L promoter regions in HCC cells were determined using qRT-PCR followed with methylated DNA immunoprecipitation (MeDIP). The results in E–G showed that knocking down TET2 rescued both upregulation of BNIP3/BNIP3L levels and decreased methylation levels of BNIP3 and BNIP3L promoters observed in HepG2-SR and Huh7-SR cells received combination therapy. Cells in E–G were cocultured with MenSCs under hypoxia for 48 h after the levels of TET2 was modulated as indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns represents not statistically significant. Full-length blots are presented in Additional file 6