Fig 1: FOXO4 plays roles in hypoxia-induced MCF-7 cells dependent on Bnip3 signal. (A) Western blot was used to determine the expression of Bnip3; (B) MCF-7 cell survival was determined using a CCK-8 assay; (C) cell death detection ELISA kit was used to detect cell apoptosis; (D) caspase-3 activity was detected using an APOPCYTO Caspase-3 Colorimetric Assay Kit. MCF-7 cells were transfected with pcDNA3.1-FOXO4 or co-transfected with pcDNA3.1-FOXO4 and pcDNA3.1-Bnip3 for 48 h and then cultured under normoxic (21% O2; 5% CO2) or under hypoxic (1% O2; 5% CO2) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the hypoxia + pcDNA3.1 group, #p < 0.05 compared with the hypoxia + pcDNA3.1-FOXO4 group.
Fig 2: Downregulation of BNIP3 reduced mitophagy, increased the production of reactive oxygen species, and promoted cell death under oxidative stress induced by SNP. (A) Relative protein expressions of LC3-I, LC3-II, and ß-actin after sh-BNIP3 transfection after SNP treatment (n = 3 per group). (B) Immunofluorescent analysis of colocalization of GFP-LC3 and mitochondria under oxidative stress after sh-Control and sh-BNIP3 transfection (n = 3 per group). (C,D) Line scans of the co-localizations between GFP-LC3 (Green) and MitoTracker (Red) for sh-Control and sh-BNIP3 under oxidative stress. (E) Flow cytometric analysis of intracellular ROS in sh-Control (a), sh-BNIP3 (b), sh-control + SNP (c), and sh-BNIP3 + SNP (d) groups (n = 3 per group). (F) Flow cytometric analysis of apoptosis in sh-Control (a), sh-BNIP3 (b), sh-control + SNP (c), and sh-BNIP3 + SNP (d) groups (n = 3 per group). (G) Statistical analysis for flow cytometric analysis of intracellular ROS. (H) Statistical analysis for flow cytometric analysis of apoptosis. NS non-significant, *p-values < 0 05, **p-value < 0.01, ***p-values < 0.001. Full-length blots are presented in Supplementary Figure 3.
Fig 3: Downregulation of GRIM-19 stimulates autophagy in murine macrophage RAW264.7 cells and THP-1-derived macrophages. (A) Representative western blotting images of GRIM-19, Beclin1, LC3B II/I and BNIP3 in RAW264.7 cells after downregulation of GRIM-19. GAPDH served as a loading control. (B) Quantification of GRIM-19, Beclin1, LC3B II/I and BNIP3 expression through relative densities. Data are shown as the mean ± SD. Three independent experiments were carried out. (C) Representative western blotting images of Grim-19, Beclin1, LC3B II/I and BNIP3 in THP-1 cells after downregulation of Grim-19. GAPDH served as a loading control. (D) Quantification of Grim-19, Beclin1, LC3B II/I and BNIP3 expression through relative densities. Data are shown as the mean ± SD. Three independent experiments were carried out. ***p < 0.001, **p < 0.01, *p < 0.05. (E) Autophagosomes were observed by TEM.
Fig 4: MnCl2 induced mitophagy by regulating ROS generation. a, b SH-SY5Y cells were treated with 200, 400, and 800 µM MnCl2 (a) and 400 µM MnCl2 with or without pretreatment with the antioxidant NAC (b) for 24 h. ROS levels were measured using a microplate reader. c BNIP3, LC3 and TOMM20 protein levels were measured by western blot after treatment with 400 µM MnCl2 with or without pretreatment with the antioxidant NAC for 24 h. ß-actin served as the internal reference. Compared with control, *P < 0.05, **P < 0.01. d Mitophagy bodies and mitochondria were observed by transmission electron microscopy (TEM) in cells treated with 400 µM MnCl2 alone or co-treated with the mitophagy inhibitor NAC for 24 h. N, nucleus. White arrow, expanded, condensed or dissolved, mitochondria aggregates. Black arrow ( ), relatively normal mitochondrial morphology. Black arrow ( ), mitophagy bodies. e After co-treatment with or without the autophagy inhibitor 3-MA, ROS levels in SH-SY5Y cells were also measured using a microplate reader
Fig 5: MicroRNA (miR)-210 overexpression ameliorated hypoxia-induced injury in PC-12 cells by down-regulating BNIP3 expression. (A) Aberrant mRNA expression of BNIP3 in PC-12 cells. (B,C) Aberrant protein expression of BNIP3 in PC-12 cells. (D–G) Effects of miR-210 overexpression on cell viability, migration, invasion and apoptosis in hypoxia-treated PC-12 cells were reversed by up-regulating BNIP3. (H,I) Western blotting analysis of apoptosis related factors. Data presented are the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. BNIP3, Bcl-2 adenovirus E1B 19 kDa-interacting protein 3; Bcl-2, mammalian B cell lymphoma-2; Bax, Bcl-2-associated X protein; C/P-, cleaved/pro; sh-NC, U6/GFP/Neo plasmid carrying a non-targeting sequence; sh-BNIP3, U6/GFP/Neo plasmid carrying sh RNA against BNIP3; pc-BNIP3, pcDNA3.1 carrying the full-length of BNIP3.
Supplier Page from Abcam for Anti-BNIP3 antibody [EPR4034]