Fig 1: Low BNIP3 expression correlates with high lipid levels and poor survival in mouse and human HCC.(A and B) Liver tumor sections from Bnip3+/+ and bnip3-/- mice at 24 weeks (A) and 40 weeks (B) of age following DEN injection, stained with H&E (top) or Oil Red O (bottom) at ×200 magnification. Scale bars, 100 µm (top left). (C) Graph of Oil Red O (ORO)–positive LD number in the liver of Bnip3+/+ (red) and bnip3-/- (blue) mice at 24 weeks (n = 12 per genotype), 32 weeks (n = 16 per genotype), and 40 weeks (n = 17 per genotype) of age following DEN injection (NS, not significant; *P < 0.05 and ***P < 0.001). (D) Comparison of relative expression of ACLY, FASN, PPARGC1A, ACADM, and BNIP3 in healthy human liver compared to HCC (14), showing fold change (log2), statistical significance (P value) of fold change, and false discovery rates (FDR) for each gene. (E and F) Overall HCC survival data obtained from the TCGA database for tumors showing BNIP3HIGH expression (above the mean, E) versus BNIP3LOW expression (below the mean, F) with either ACACALOW or ACACAHIGH expression. (G) H&E-stained sections of human BNIP3HIGH-expressing HCC (top) or BNIP3LOW-expressing HCC (bottom), with either ACACALOW (left)– or ACACAHIGH (right)–expressing HCC. Scale bars, 10 µm.
Fig 2: Loss of BNIP3 promotes HCC initiation and tumor growth.(A) Representative images of HCC tumors forming in the liver of Bnip3+/+ and bnip3-/- mice at 24 and 40 weeks of age following DEN injection. Scale bars, 12.7 mm. (B) Graph of tumor number forming in the liver of Bnip3+/+ (red) and bnip3-/- (blue) mice at 24 weeks (n = 9 per genotype), 32 weeks (n = 14 per genotype), and 40 weeks (n = 13 per genotype) of age following DEN injection. (NS, not significant; **P < 0.01). (C) Graph of tumor size in the liver of Bnip3+/+ (red) and bnip3-/- (blue) mice at 24 weeks (n = 9 per genotype), 32 weeks (n = 14 per genotype), and 40 weeks (n = 13 per genotype) of age following DEN injection (NS, not significant; ** P < 0.01). Scale bars, 100 µm (top left). (D) Liver sections from Bnip3+/+ mice at 24, 32, and 40 weeks of age following DEN injection, stained with H&E (top row), Ki67 (middle row), and BNIP3 (bottom row). (E) Quantification of immunohistochemical staining for BNIP3 on liver sections from Bnip3+/+ mice at 24 weeks (n = 5), 32 weeks (n = 7), and 40 weeks (n = 5) of age following DEN injection (**P < 0.05, ***P < 0.005, and ****P < 0.0005). (F) qPCR for Bnip3 mRNA isolated from HCC lesions and adjacent normal liver in Bnip3+/+ mice at 32 weeks (n = 12) and 40 weeks (n = 12) of age following DEN injection (****P < 0.0001). (G) Western blot for BNIP3 in protein lysates isolated from HCC tumor lesions (T) and adjacent normal (N) liver in Bnip3+/+ mice at 32 and 40 weeks of age following DEN injection.
Fig 3: BNIP3 promotes colocalization of LDs, mitochondria, and lysosomes in mitolipophagy.(A to C) Superresolution immunofluorescence microscopy of bnip3-/- HCC cells reconstituted with EV (A), HA-BNIP3WT (B), or HA-BNIP3W18A (C) grown for 24 hours in 50 µM LALi + 6 hours in 100 µM oleic acid and then stained with BODIPY 493/503 (red), TOMM20 (magenta), and LAMP1 (green) to visualize overlap between LDs (green), mitochondria (magenta), and lysosomes (magenta). Two representative high-magnification images (highlighted by white dashed square, left) are shown for each in (A), (B), and (C). Linescan analysis of colocalization of LDs (red) with mitochondria (magenta) and lysosomes (green) is shown for each of the two representative high-magnification images from bnip3-/- HCC cells reconstituted with EV (A), HA-BNIP3WT (B), or HA-BNIP3W18A (C), illustrating that only BNIP3WT promoted LD-lysosome overlap. (D) Graphical representation of model proposed.
Fig 4: BNIP3 promotes lipophagy, and inhibiting lysosomal lipase limits HCC cell growth.(A) Fluorescent microscopy imaging of BODIPY 493/503 staining in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A ± 50 µM LALi + 100 µM oleic acid. (B) Graph of BODIPY 493/503 staining in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A ± 50 µM LALi + 100 µM oleic acid (ns, not significant; **P < 0.01 and ****P < 0.0001; at least 20 cells per field, each data point is one field, n = 10 fields per condition). (C) ImageStream flow cytometric quantification of LDs in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A grown for 24 hours in 50 µM LALi + 6 hours in 100 µM oleic acid and then released into lipid free–media for 0, 8, and 16 hours. At least 10,000 cells per condition were quantified for n = 2 experiments. (D) Rate of cell growth determined by IncuCyte of bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A ± 50 µM LALi in n = 3 experiments for each condition. (E to G) OCR (166 µM palmitate and 2 mM d-glucose) in bnip3-/- HCC cells reconstituted with EV (E), HA-BNIP3WT (F), or HA-BNIP3W18A. Cells were plated 12 times per plate for each time point per genotype per experiment for n = 3 experiments. (G) ±50 µM LALi. (H and I) LC-MS analysis of triglycerides and phosphoglycerols decreased (fold change > 2.0 and P > 0.05) in bnip3-/- HCC cells expressing BNIP3 WT compared to EV (blue bars) and increased by 24-hour treatment with 50 µM LALi (red bars). Lipidomics were performed on five biological replicates per condition.
Fig 5: BNIP3-dependent mitophagy suppresses mitochondrial mass, glucose oxidation, and HCC cell growth.(A) Western blot for a-HA and a-AFP in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A. (B) Pulldown for GFP-LC3 in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A in the presence (+)/absence (-) of 100 nM bafilomycin A1 and blotted for GFP (LC3) or HA (BNIP3) in input (left) or pulldown lysates (right). (C) Immunofluorescent staining for TOMM20 and LC3 in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A in the presence of 100 nM bafilomycin A1. Yellow arrows in (v), (viii), and (xi) point to representative fragmented mitochondria staining positive for TOMM20 and LC3B. (D) Quantification of LC3-positive puncta (magenta) overlapping (white) with TOMM20 staining (green) per cell in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A in the presence (+) or absence (-) of 100 nM bafilomycin A1 (NS, not significant; ****P < 0.0001; at least five cells per field, each data point is one field, n = 10 fields per condition). (E) Western blot analysis for PDHA, LC3B, and p62/Sqstm1 in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A in the presence (+)/absence (-) of 100 nM bafilomycin A1. (F) qPCR for mt:nuc DNA ratio in bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A (NS, not significant; *** P < 0.001 and ****P < 0.0001). Experiments performed 12 times per condition, n = 2 experiments. (G) OCR (25 mM d-glucose and 2 mM l-glutamine) of bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A. Cells were plated 12 times per plate/time point/genotype/experiment, n = 3 experiments. (H) Rate of cell growth determined by IncuCyte of bnip3-/- HCC cells reconstituted with EV, HA-BNIP3WT, or HA-BNIP3W18A, n = 3 experiments.
Supplier Page from MilliporeSigma for Anti-BNIP3 antibody produced in rabbit