Fig. 1

BNIP3 and BNIP3L levels are negatively correlated with sorafenib resistance in HCC-SR cells. A Heatmap of BNIP3 and BNIP3L mRNA expression in HepG2 in response to coculture with MenSCs for 72 h. B Half maximal inhibitory concentration (IC50) at 48 h was determined using CCK8 analysis (left, Mean, n = 6). Statistical analysis of relative IC 50 levels (right). C The levels of BNIP3, BNIP3L, and β-actin were determined using immunoblotting analysis 48 h after cells were cultured under normoxia or hypoxia (1%O₂) as indicated. D Transcription levels of BNIP3 and BNIP3L in HCC-SR cells were semi-quantified by RT-PCR analysis (27 PCR amplification cycles in HepG2-SR and Huh7-SR, 30 PCR amplification cycles in HCCLM3-SR). Cells were treated with 5 µM 5-aza-2-deoxycytidine (5-Aza) for 1 week and cultured under hypoxia for 48 h before harvest. Negative control (NC) group: treated without 5-Aza, other cultural conditions were the same as the 5-Aza group. E The 5-mc levels of BNIP3 and BNIP3L promoter regions in HCC cells were determined using qRT-PCR followed with methylated DNA immunoprecipitation (MeDIP). F Cell viability was determined using CCK-8 analysis 48 h after cells were treated as indicated. Cells were treated with 10 µM sorafenib under hypoxia after the levels of BNIP3 or BNIP3L were modulated as indicated. G Proliferation ability of HCC-SR cells was determined using clone formation assay. Cells were cultured in complete DMEM medium containing 6 µM sorafenib for 1 week after the levels of BNIP3 or BNIP3L were modulated as indicated. H Cell viability of HCC-SR cells was determined using Annexin V/PI analysis. Cells were treated with 10 µM sorafenib for 48 h under hypoxia after the levels of BNIP3 or BNIP3L were modulated as indicated. ***p < 0.001, ****p < 0.0001. ns represents not statistically significant. Full-length blots/gels are presented in Additional file 6