Fig 1: Ethanol-induced expression of BNIP3 and NIX was abolished in primary cultured HIF-1β knockout mouse hepatocytes.(A) Primary cultured wild type and HIF-1β knockout hepatocytes were treated with ethanol (80 mM) for 6 hours. mRNA was isolated from cultured hepatocytes and real-time RT-PCR was performed as described in the Materials and Methods. Data are presented as means ± SE (n = 4). * p<0.05. One way ANOVA with Scheffé's post hoc test. (B) Cells were treated as in (A), total cell lysates were subjected to western blot analysis. Densitometry was performed and data are presented as a ratio vs HIF-1β, Alb Cre- control (n = 3).
Fig 2: Changes in mitophagy levels in AT-II cells of rats with bronchopulmonary dysplasia. (A) Representative immunostaining of AT-II cell fluorescence double staining (x800 magnification; scale bar, 25 μm). Green represents the mitochondria marker (COX4), red represents autophagosome marker (LC3B) and double staining is shown in yellow. (B) Line scan data of fluorescence intensity in the corresponding images to show the degree of co-localisation of COX4 and Lc3B using ImageJ software1.80. (C) Western blot analyses of NIX and COX4 in the lungs in the C group and M group. density analyses of (D) NIX and (E) COX4. Blots were stripped and reblotted using an anti-β-actin antibody as a loading control. (F) Correlation analyses between radial alveolar counts and NIX mediated mitophagy relative expression in the rat lungs. NIX-mediated mitophagy was negatively correlated with alveolar development index (r=-0.32; P<0.05). data represent at least six independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 vs. control. AT-II, alveolar type II cells; COX4, cytochrome C oxidase subunit IV isoform I; LC3B, microtubule-associated protein-1 light chain-3B; NIX, Nip3-like protein X; C, control; M, model.
Fig 3: Acute ethanol treatment increased expression of BNIP3 and NIX in primary hepatocytes and mouse liver.Primary cultured mouse hepatocytes were treated with ethanol (80 mM) for 6 hours. Densitometry was performed and data are presented as a ratio vs control (n = 3). (A) Total cell lysates from cultured hepatocytes were subjected to western blot analysis. (B) Male C57BL/6 mice were treated with either water or ethanol (4.5 g/kg) by gavage for 6, 12 or 16 hours. Hepatic mRNA was isolated and real-time RT-PCR was performed as described in the Materials and Methods. Data are presented as means ± SE (n = 4–6). * p<0.05, vs 0 hour control. One way ANOVA with Scheffé's post hoc test. (C) Mice were treated as in (B) and total liver lysates from the mice (16 hours treatment) were subjected to western blot analysis. Densitometry was performed and data are presented as a ratio of control (n = 3).
Fig 4: Berberine (BBR) regulates BNIP3-mediated mitochondrial autophagy to attenuate ischemia/reperfusion (I/R) injury in myocardial ischemia-reperfusion injury (MIRI) model rats. After transfection with shBNIP3/shNC, rats were administered BBR orally (300 mg/kg, once a day for 3 consecutive days) or not and then exposed to I/R. (A, B) Western blotting was used to detect the protein expression of LC3, NIX, and P62; GAPDH was used as a control. (C) After I/R, 2,3,5-triphenyl tetrazolium (TTC) staining (10×) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (400×) was performed on myocardial tissues from each group. (D, E) Statistical analysis of the infarct size and TUNEL index. 1, sham group; 2, I/R group; 3, BBR+I/R group; 4, shNC+BBR+ I/R group; 5,shHIF-1α+BBR+I/R group. The data are the means ± SD (n=3). *P < 0.05 compared to the sham group; #P < 0.05 compared to the I/R group; &P < 0.05 compared to the BBR+I/R group.
Fig 5: TP53INP1 deficiency is linked with impaired PINK/PARKIN mitophagy After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin. HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control. Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin. Data information: Results are representative of three independent experiments. Source data are available online for this figure.
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