Fig 2: JNK-ERK AMPK pathway was involved in the protective effect of pinocembrin which activated BNIP3-mediated mitophagy. BV2 cells were pretreated with SP600125 (JNK inhibitor, 10 µM) or FR 108204 (ERK inhibitor, 10 µM) for 30 min before the pinocembrin administration. Pinocembrin was added to the culture medium for 1 h before exposing to NA (normal air) or IH (intermittent hypoxia). a Total proteins from the microglia were immediately separated by SDS-PAGE gel and analyzed by western blot. Blots were probed with specific primary antibodies as indicated. b Quantitation of pinocembrin-activated ERK1/2 and JNK MAPK pathways. Depicted is the weighted average density of bands (p-JNK and p-ERK) normalized to JNK or ERK at 24-h exposure with NA or IH, respectively. c Proliferation of BV2 cells in different groups was analyzed by CCK-8 assay. d Flow cytometric analysis of microglia for mtROS with MitoSOX. e Western analysis showed pinocembrin induced Bcl-2 expression under IH exposure, but reduced by JNK or ERK inhibitor. GAPDH was shown as a loading control. f Representative images of NLRP3 (red) and ASC (green) by immunofluorescent staining of BV2 cells. Data are representative of at least 3 independent experiments and presented as mean ± SEM. †p < 0.05 vs. the NA group; #p < 0.05 vs. the IH group; *p < 0.05 vs. the IH + PIN group

Fig 3: Gestational exposure to environmental stress triggers BNIP3-dependent mitophagy via activating GCN2/ATF4 signaling in human placental trophoblasts. (A–D) Human JEG-3 cells were treated with Cd for 0, 2, 6 or 12 h (n = 3 per group). (A) Representative immunoblots of GCN2, p-eIF2α/eIF2α and ATF4 proteins in the cells. (B–D) Quantification for GCN2, p-eIF2α and ATF4. (E–L) Human JEG-3 cells were pretreated with GCN2 siR before Cd stimulation (n = 3 per group). (E) P4 in the medium. (F) Representative immunoblots of BNIP3, LC3BI/II, HSP60, COX IV and CYP11A1 proteins in the cells. (G–I) Quantification for BNIP3, LC3BI/II, HSP60, COX IV and CYP11A1. (J) Representative immunoblots of ATF4 and GCN2 proteins in the cells. (K and L) Quantification ATF4 for and GCN2. Data are expressed as the mean ± SEM. Multiple comparisons were performed using ANOVA. The post hoc test is executed adopting Bonferroni or Tamhane's T2 method following the result of homogeneity of variance test. *P < 0.05, **P < 0.01 versus PBS group, #P < 0.05, ##P < 0.01 versus Cd group.

Fig 4: Bnip3 deficiency accumulates damaged mitochondria and ROS production following renal IR.Bnip3-KO and WT mice were subjected to renal IR or sham operation (sham). Renal cortex was fixed and processed for TEM analysis and DHE staining. a Representative TEM images of mitochondrial morphology in proximal tubular cells. b Percentage of abnormal mitochondria (swollen with evidence of severely disrupted cristae over all mitochondria) (n = 4). c Representative images of DHE staining. DHE nuclear staining indicates the presence of reactive oxygen species (ROS) (n = 4). Scale bar: 100 µm. d Quantification of DHE fluorescence intensity. Each symbol (circle and diamond) represents an individual mouse. Error bars: SEM. **p < 0.01; ***p < 0.001

Fig 5: BNIP3 expression is regulated by p38 and JNK under hypoxia.a HaCaT cells were exposed to hypoxia (2%) and incubated for the indicated times, and total proteins were harvested for detection of the expression of BNIP3 and the activities of p38/MAPK and JNK/MAPK. HIF-1a was used as an indicator of hypoxia. GAPDH was used as the loading control. b–e HaCaT cells were exposed to hypoxia (2%) for 6 h. The SB203580 (SB, 5 µM) and SP600125 (SP, 5 µM) MAPK inhibitors were added 1 h before hypoxia exposure. b The extracted proteins were then immunoblotted with the indicated antibodies. HIF-1a was used as an indicator of hypoxia. GAPDH was used as the loading control. Fluorescence staining of BNIP3 (c), and LC3 expression (d) in HaCaT cells are shown. Nuclei were stained with DAPI. Scale bar = 25 µm. e Wound-healing assays and f single-cell motility assays were performed using the indicated cells. Representative images of wound healing, including keratinocyte trajectories. Scale bar = 100 µm. The results of quantitative analysis (g) are represented by the mean ± SEM (n = 3). *P < 0.05 vs. Norm group, and #P < 0.05 vs. Hypo group