Fig 2: Fractionation procedure and verification. A Schematic overview of MAM and mitochondrial enrichment by differential centrifugation after homogenizing whole brains from control of CSD-exposed mice. ER, endoplasmic reticulum; CM, crude mitochondria; PM, pure mitochondria; MAM, mitochondria-associated membranes; S, supernatant; P, pellet. B Verification of enriched cellular fractions by immunoblot against VDAC1, MFN2, calnexin, GRP75/mortalin, COXI (cytochrome c oxidase subunit 1), and COXIV (cytochrome c oxidase subunit IV). C Validation subcellular fractioning: as expected, the mitochondrial MAM markers VDAC1, GRP75 and MFN2 levels are most abundent in the pure mitochondrial fraction but were also increased in the MAM fraction (in comparison to ER). In contrast, calnexin, a MAM marker from the ER, was most abundant in the ER fraction, but was also increased in the MAM fraction (as compared to the pure mitochondrial fraction). Bars show the mean ± SEM with each data point representing one mouse

Fig 3: Mitochondria are fragmented in CDDP resistant cells.A Images of mitochondrial network in sensitive (2008, U2OS) and resistant (C13, U2OS-PT) cells acquired by confocal microscopy Zeiss using TOM20 immunostaining, 488. Scale bar 20 µm. B Mitochondria segmentation was performed using the ImageJ Squassh plugin (Rizk et al., 2014); size and morphology features were measured by using Fiji. Data from 15 different cells per cell line. *p < 0.05, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells. C Images of mitochondrial morphology in sensitive (2008, U2OS) and resistant (C13, U2OS-PT) cells acquired by Tecnai G2 (FEI) transmission electron microscope operating at 100 kV; images were collected by a F114 (TVIPS) CCD camera. The TEM images and experiment are performed by the University of Padova electron microscopy facility. Scale bar 1 µm and 500 nm. D The morphometric analysis was performed using ImageJ freehand tool (at least 5 cells per sample, at least 50 images/sample). Data are the mean ± SEM of three different experiments; **p < 0.01, ***p < 0.001, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells. E, F Expression of OPA1, MFN1, MFN2, MFFs, p-DRP1 and total DRP1. G The optical density (O.D.) was normalized respectively to TOM20; for total DRP1 to ß-ACTIN for 2008-C13 or calnexin for U2OS-U2OS-PT in cancer cells sensitive and resistant. The data are expressed as ratio of resistant cells to sensitive. Data are the mean ± SEM of 4–5 different experiments; *p < 0.05; **p < 0.01, ***p < 0.001, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells. H mRNA expression of genes OPA1, MFN2, DRP1 and H-FIS1 normalized to ß-actin for 2008-C13 or calnexin for U2OS-U2OS-PT. The data are expressed as a ratio of resistant cells to sensitive cells set to 1. Data are the mean ± SEM of 4–5 different experiments; *p < 0.05; **p < 0.01, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells.

Fig 4: Lung adenocarcinoma patients with high OPA1 and DRP1 mRNA levels treated with chemotherapy display a worse prognosis.Kaplan–Meier survival curves of lung adenocarcinoma patients in PanCancer Atlas. Red and blue lines represent survival curves of patients with lung tumor tissues with high and low expression levels of OPA1 (A) MFN1 (B), MFN2 (C), and DRP1 (D) mRNA. The cutoff value was determined as the quantile value of the expression levels of each mRNA (n = 120). Dashed lines indicate 95% confidence intervals. The p values were calculated using a log-rank test. The hazard ratio was calculated with Cox PH Model. HR hazard ratio.