Fig 2: Schematic diagram of mitochondrial dynamics and biogenesis associated with MG. Decreased Mfn1/2, Opa1, Fis1 and Drp1 proteins leads to imbalance of mitochondrial fusion and fission, affecting mitochondrial structure and regulation of mitochondrial permeability transition pore channels. AMPK modulates activation of PGC-1α and binding to NRF-1 to activate TFAM. Decreased expression of these proteins decreases mitochondrial biosynthesis, energy generation and ATP synthesis, affecting the binding of myosin and actin and causing MG symptoms, such as limb weakness, eyelid ptosis, chewing weakness and dysphagia. MG, myasthenia gravis; Mfn1/2, mitofusion1/2; Opa1, optic atrophy type 1; Drp1, dynamin-related protein 1; Fis1, fission 1; AMPK, AMP-activated protein kinase; PGC-1α, peroxisome proliferators activated receptor γ coactivator 1α; NRF-1, nuclear respiratory factor-1; TFAM, mitochondrial transcription factor A; mPTP, mitochondrial permeability transition pore.

Fig 3: Differential gene expression in the kidney of the wild-type and ΔMtln-1 knockout mice. (a) Volcano plot of differentially expressed genes. X-axis corresponds to the log-scale fold change of expression, ΔMtln-1, relative to the wild-type, while the y-axis corresponds to the p-value. (b) Gene set enrichment analysis result demonstrating downregulation of genes from the category of fatty acids metabolism (left panel) and the category of oxidative phosphorylation (right panel) in the kidneys of ΔMtln-1 mice. (c) Immunoblotting of kidney extracts of the wild-type (left 3 lanes) and ΔMtln-1 (right 3 lanes) mice. Antibodies against mitochondrial proteins Cyb5r3, Opa1, Mfn2, Vdac1, and control antibodies against Gapdh were used as indicated left to the panels.

Fig 4: Mitochondrial respiration in Opa1−/− and Opa1+/+ MEF. (a) Oxygen consumption rates were measured in both Opa1−/− and Opa1+/+ MEFs with the Seahorse XFe96 extracellular flux analyzer. The OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). (b) Basal respiration (R) represents the mitochondrial respiration sustained by endogenous substrates. Non-phosphorylating respiration (O) represents the residual respiration in the presence of oligomycin, whereas the maximal uncoupled stimulated respiration (F) was determined by titration of FCCP (0.25–3 μM). Values are expressed as respiratory control ratios, which in each case are inferred from the maximal uncoupled stimulated respiration (F). Histograms show the mean ± S.D values of four independent experiments. (c) NAD/NADH ratio and ATP in Opa1−/− and Opa1+/+ MEFs incubated in glucose medium. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s unpaired t‐test (**p-value < 0.01).

Fig 5: TIM-4 interacts with ANXA2 to regulate the PI3K/AKT signaling pathway and mitochondrial function.A Venn diagram depicting the comparison of proteins identified in A549-LV-CON, A549-LV-TIM-4, and HEK-293 cells. The shared proteins were excluded from the differential lists. B, C Co-immunoprecipitation (Co-IP) assays were performed in A549 and H23 cells overexpressing TIM-4 or control. D, E OCR, mitochondrial basal respiration and maximal respiration of A549 cells and H23 cells transfected with siRNA of ANXA2 or control. F Mitochondrial membrane potential (JC-1 aggregates) in A549 cells transfected with siRNA targeting ANXA2 were accessed by the JC-1 assay kit according to the manufacture’s instruction. G Mitochondria fitness was tested with Mito-tracker Deep Red. H Flow cytometry dot plots showed the percentage of depolarized mitochondria in A549 cell lines transfected with siRNA targeting ANXA2. I Western blotting analysis of OPA1, p-AKT, PCNA, and ANXA2 expression in siANXA2-transfected lung cancer cell lines. J Flow cytometry histograms showing the level of Ki67 in lung cancer cell lines transfected with siRNA targeting ANXA2. Three independent experiments were conducted for each result and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed unpaired t test) for (D), (E), (F–H), and (J). ns means non-significance; *P < 0.05; **P < 0.01; ***P < 0.001.