Fig. 7: Mdivi-1 inhibited mitophagy and exacerbated mtDNA-mediated inflammation in M9a+shEPAS1 cells during hypoxia. | Cell Death Discovery

Fig. 7: Mdivi-1 inhibited mitophagy and exacerbated mtDNA-mediated inflammation in M9a+shEPAS1 cells during hypoxia.

From: Mitochondrial retrograde signaling initiates HIF-1α/BNIP3/NIX-mediated mitophagy in Tibetan high-altitude adaptation

Fig. 7: Mdivi-1 inhibited mitophagy and exacerbated mtDNA-mediated inflammation in M9a+shEPAS1 cells during hypoxia.The alt text for this image may have been generated using AI.

A Schematic diagram of the mechanism of action of Mdivi-1. B Western blot analysis of DRP1, NIX, BNIP3, LC3B, and p62 protein levels in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. C Autophagic flux was assessed by quantifying the colocalization of LC3B (green) and Mitotracker (red) (mitophagosomes) using immunofluorescence in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. DAPI (blue) stains the nucleus. Scale bar: 20 μm. D–F Mitochondrial OCR (basal, ATP-linked, maximal), MMP, and ATP content were measured in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. G CCK-8 assay showing cell viability in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. H Cell apoptosis was determined in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia using Annexin V-FITC staining. I Western blot analysis of C-Caspase-3 and BAX protein levels in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. J Cytoplasmic free mtDNA was determined by staining dsDNA (green) and Mitotracker (red) using immunofluorescence in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. DAPI (blue) stains the nucleus. dsDNA that does not colocalize with either the nucleus or mitochondria is considered cytoplasmic free mtDNA. Scale bar: 20 μm. K Western blot analysis of NLRP3, IL1β, cGAS, and STING protein levels in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. L Inflammatory cytokines IL6, IL1β, and TNF-α in the culture medium were measured using ELISA kits in M9a+shEPAS1 cells after Mdivi-1 treatment under hypoxia. M9a+shEPAS1 cells (n = 4) were treated with 50 μM Mdivi-1 or DMSO (pre-treatment for 4 h, followed by treatment for 48 h) under hypoxia (1% O₂).#1–4 represent individual samples. Data are presented as the mean ± SD of three independent experiments. Two-tailed, paired Student’s t tests; P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

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