Fig 2: Mdivi-1 ameliorates mitochondrial morphology and autophagy dysregulation and also restores respiratory chain complex protein expression in F594L mutant cells.(A) Western blot analysis for OPA1, DRP1, and p-DRP1 levels following treatment with MYLS22, GSK3-IN-3, and Mdivi-1 examining the modulation of these proteins under different pharmacological conditions. Data quantification below the blots shows the relative protein levels normalized to ACTIN. All genes come from the same samples run on different, but concurrent, blots, except for OPA1, which was run on a separate occasion. (B) Confocal microscopy illustrated mitochondrial morphology across various conditions: control cells, F594L mutants, F594L mutants with OPA1 lentiviral transduction, and F594L mutants treated with Mdivi-1; all were stained with MitoTracker. The accompanying quantification bar graph shows the proportion of cells exhibiting filamentous, intermediate, or fragmented mitochondria. (C) Detailed confocal images showing mitochondrial (red) and lysosomal (green) colocalization. They depict the levels of mitophagy, and zoomed-in views are also provided. (D) Western blot analysis was used to quantify the levels of OPA1 and LC3 as well as to assess an increased ratio of L-OPA1/S-OPA1 isoforms, across different treatment groups. The results are derived from the same samples run on different but concurrent blots. (E) Western blot analysis of mitochondrial respiratory chain complexes, including CV-ATP5A, CIII-UQCRC2, CII-SDHB, CIV-CO2, and CI-NDUFB8. Data quantification indicated the effects of OPA1 overexpression and Mdivi-1 treatment on the levels of complex expression. The results are derived from the same samples run on different but concurrent blots. Statistical analysis was by 1-way ANOVA and Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 (A, D, and E). Scale bars: 20 μm (B), 20 μm (inset, 4 μm; C).

Fig 3: Effect of chronic galactose treatment on gene and protein expressions.Myotubes were grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose. Total RNA was isolated from the cells and analyzed by qPCR, while protein samples were harvested analyzed as described in Materials and Methods. Gene expressions were normalized to 36B4 and protein expressions to β-actin, except phosphorylated AMP-activated protein kinase (p-AMPK), which were normalized to total AMPK. Values in A and B represent fold change of genes/proteins in galactose-treated myotubes relative to glucose-treated myotubes, given as means ± SEM (n = 5). (A) Genes analyzed; CPT1b, carnitine palmitoyltransferase-1b; CYC1, cytochrome C; MCAD, acyl-coenzyme A-dehydrogenase; MYH2, myosin heavy chain 2, SLC2A1 and SLC2A4; glucose transporter 1 and 4, HKII; hexokinase II, GALK1 and 2; galactokinase 1 and 2, GALT; galactose-1-phosphate uridylyltransferase, PDK4; pyruvate dehydrogenase kinase 4. (B) Protein expression of myosin, ATP synthase subunit, slow muscle, complex II subunit, complex IV subunit II, complex I subunit NDUFB8, PDK4; pyruvate dehydrogenase 4, P-AMPK and AMPK. (C) Representative corresponding Western blots.

Fig 4: ONC201/TIC10 causes loss of mitochondrial proteins and activation of stress response in everolimus sensitive and resistant cells.(A) CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with ONC201/TIC10 at the indicated concentrations, and cell lysates were immunoblotted for TUFM, TFAM, BiP, ATF4, CHOP, and OXPHOS complexes (Complex I subunit NDUFB8, Complex II subunit 30 kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha), and β-actin. Quantitation of TUFM, TFAM, BiP, ATF4, and CHOP using ImageJ analysis. Protein expression levels were normalized to β-actin and are shown as average of two replicates ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001. (B) CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with everolimus, ONC201/TIC10, and combination at the indicated concentrations, and cell lysates were immunoblotted for the same proteins as above. Figure 4—source data 1.Original files of the full blot images for Figure 4A. Figure 4—source data 2.Original files of the full blot images for Figure 4B. Figure 4—source data 3.Figures with uncropped blot images for Figure 4A, B.

Fig 5: HTyr and OPWPs extracts affect mitochondrial functions. (A) Representative immunoblots of lysates of HCT116 and LoVo cells untreated or treated with HTyr or OPWPs extracts for 72 h for assessing the proteins ATP5A, UQCRC2, SDHB, COX II, and NDUFB8 belonging to the respiratory chain complexes V, III, II, IV, and I, respectively. The histograms report the overall OXPHOS protein quantification normalized to β-Actin or α-Tubulin, used as loading controls, and show the mean ± SD of three independent experiments. (B) ATP levels measured in HCT116 and LoVo cells treated as in (A) and expressed as picomoles (pmol). (C) Flow cytometry analysis of the cells treated as in (A) and stained with TMRE to evaluate the mitochondrial membrane potential calculated as Median Fluorescence Intensity (MFI) of two independent experiments. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001 (ANOVA with Dunnett’s post-test).