Fig. 4

Combination therapy switches protective autophagy to autophagic cell death in Huh7-SR cells. A The levels of BNIP3, BNIP3L, LC3, and β-actin were determined using immunoblotting analysis 48 h after cells were treated as indicated. The ratio of LC3-II/LC3-I was labelled below the LC3 lane. BafA1 was added 12 h before harvest. B, C The expression of mCherry-GFP-LC3 was determined by confocal microscopy analysis 48 h after cells were treated as indicated. mCherry⁺ GFP⁻ and mCherry⁺ GFP⁺ puncta were quantified and summarized to determine the relative autophagy flux. Scale bar: 5 µm. D The levels of BNIP3, BNIP3L, LC3, and β-actin in Huh7-SR cells were determined using immunoblotting analysis. Cells were administered sorafenib or combination therapy under hypoxia for 48 h after the levels of BNIP3 and BNIP3L were modulated as indicated. BafA1 was added 12 h before harvest. E The expression of GFP-LC3 was determined by confocal microscopy analysis. Cells were administered combination therapy under hypoxia for 48 h. Scale bar: 5 µm. F The mRNA levels of BNIP3 and BNIP3L were determined using qRT-PCR 48 h after cells were treated as indicated. G The levels of BNIP3, BNIP3L, and β-actin were determined using immunoblotting analysis 48 h after Huh7-SR cells were treated as indicated. Cells were treated with MG132 (10 μM) or CQ (20 μM) 6 h before harvest. H The levels of LC3 and β-actin were determined using immunoblotting analysis 48 h after Huh7-SR cells were treated as indicated. I Cell viability was determined using CCK-8 analysis 48 h after Huh7-SR cells were treated as indicated. 3-MA was added 24 h before harvest. J–K The interactions between BCL2 and Beclin1/BNIP3/BNIP3L (J) or Beclin1 and ATG14/VPS34 (K) in Huh7-SR cells were determined using immunoblotting analysis followed with co-IP 48 h after indicated treatments. *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.