Fig 2: High insulin level inhibited BHB-induced protection on mitochondrial function in HK2 cells under low glucose or hypoxic conditions through SMCT1 in HK2 cells. (A, D) Validation of overexpression of SMCT1; (B, C, E, F) Expression of PGC1α, Mfn1, and Drp1 in HK2 cells under overexpression of SMCT1; (G–I) ATP production in different groups. In panels (A, C, G, H) ap < 0.05, compared with the LG + BHB + HI group (control group); bp < 0.05, compared with the LG + BHB + HI + AAV-SMCT1 NC group (vector group); in panels (D, F, I) ap < 0.05, compared with Hypoxic + BHB + HI group; bp < 0.05, compared with the Hypoxic +BHB + HI + AAV-SMCT1 NC group.

Fig 3: The relationship between NOTCH1 insufficiency and mitochondrial dysfunction in BAV-TAA.(a) H and E staining of non-diseased and BAV-TAA aortic tissues. The scale bar represents 1 mm in the low field and 200 μm in the high field. (b) Representative images of the western blotting analysis of the expression of NOTCH1, the mitochondrial dynamics proteins DRP-1, MFF, MFN1, and MFN2 and the contractile phenotype proteins SM22 and CNN1 in six non-diseased and six BAV-TAA aortic tissue fragments. (c) Quantification of the total band densities of the proteins normalized to the corresponding band density of β-actin (n = 6, *p < 0.05, two‐tailed Student’s t tests were used between two groups). (d) Correlation analysis among the quantified protein expression levels of NOTCH1, MFN1, and MFN2. A positive correlation was found between NOTCH1 and MFN1 or MFN2 in aortic tissues. The R2 between NOTCH1 and MFN2 was 0.8069, and that between NOTCH1 and MFN1 was 0.6137. (e) Correlation analysis between the quantified protein expression of NOTCH1 and DRP1 or MFF. No correlation was found. (f) The enriched canonical pathways identified by IPA. (g) Heatmap of the expression of enriched proteins involved in mitochondrial dysfunction, the oxidative phosphorylation pathways and acute phase response signaling. (h) Volcano plot visualization of the differentially expressed proteins related to mitochondrial dysfunction. The colors indicate the following: gray, no differential expression; red, upregulated proteins; and blue, downregulated proteins. The proteins related to mitochondrial dysfunction are labeled. All the data are expressed as the means ± SDs. Figure 1—source data 1.The original raw data for western blotting. Figure 1—source data 2.List of total differential protein expression between non-diseased and BAV-TAA aortic tissues. Figure 1—source data 3.The total enriched canonical pathways in BAV-TAA aortic tissues. Figure 1—source data 4.List of total protein expression between non-diseased and BAV-TAA aortic tissues.

Fig 4: MFN1/2 promotes erastin-induced ferroptosis. (A) Immunoprecipitation assay of the interaction between STING1, MFN1, MFN2, and OPA1 in mitochondria fractions of PANC1 cells with or without erastin (10 μM) treatment for 12 h. (B) Western blot analysis of MFN1 and MFN2 expression in control and MFN1/2KD PANC1 cells. (C–F) Indicated PANC1 cells were treated with erastin (10 μM, 24 h) or zalcitabine (20 μM, 72 h), and then cell death, intracellular mtROS, intracellular lipid peroxidation, and extracellular HMGB1 were assayed (n = 3 wells/group; ANOVA with Tukey’s multiple comparisons test; *P < 0.05 versus control shRNA group). (G) Indicated MEFs cells were treated with erastin (5 μM, 24 h) or zalcitabine (20 μM, 72 h) and then cell death was assayed (n = 3 wells/group; ANOVA with Tukey’s multiple comparisons test; *P < 0.05 versus WT group). (H) Indicated PANC1 cells were treated with erastin (10 μM) for 24 h and then cell death was assayed (n = 3 wells/group; ANOVA with Tukey’s multiple comparisons test; *P < 0.05 versus control shRNA group). Data are from two or three independent experiments.

Fig 5: The critical role of NME3 in maintaining mitochondrial morphology. (A) Representative images of mitochondrial morphology of HeLa cells expressing shLacZ or shNME3 are shown. Cells were fixed for COX4 IF staining as a mitochondrial marker. (Scale bar, 10 μm.) Cells that contain >75% of total mitochondria in tubular or fragmented organization were categorized and the rest of the population was defined as cells with mitochondria of intermediate morphology. Mitochondrial morphology in tubular, intermediate, and fragmented were counted from 100 cells (n = 3). Data are presented as mean ± SEM. * p < 0.05, *** p < 0.005 compared with that of shLacZ by Student’s t test. (B) HeLa cells expressing shLacZ or shNME3 were harvested for Western blotting using antibodies against ATF4, MFN1/2, DRP1, Mid49/51, SOD2, and β-tubulin. (C,D) shLacZ and shNME3 cells were transfected with GFP-NME3 variants including wild-type (WT) and N-terminal deleted-mutant (ΔN) for 8 h and then fixed for mitochondrial morphology analysis. (C) Representative images of COX4 staining. (Scale bar, 10 μm.) (D) Quantitation results of mitochondrial morphology were analyzed from 100 cells (n = 3). Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.005 compared with that of shNME3 based on Student’s t test.