Fig 2: Induction of MFN2 via leflunomide replicates MARCH5 loss and triggers anti-MM responses. A Western blot analysis of MFN2 was performed in AMO cells, 72 h after treatment with increasing doses of Leflunomide or DMSO as vehicle; GAPDH was used as loading control. B Heatmap shows cell viability assessed by Cell Titer Glo assay in MM cells treated with Leflunomide for 48 h. Viable cells are reported as percentage of vehicle. C Cell viability assessed by Cell Titer Glo assay in AMO and H929 cells treated with Leflunomide for 72 h, and then co-cultured with HS5 cells for 48 h. Viable cells were expressed as the percentage of vehicle. Data represent the average ± SD of three independent experiments. *p < 0.05. D Western blot analysis of NDUFS1, NDUFAB1, and COX4 expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. *p < 0.05. F Western blot analysis of PUMA and NOXA expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. G In vivo tumor growth evaluation of subcutaneous AMO xenograft receiving i.p. Leflunomide (20 mg/kg), or vehicle as control; administrations were performed 5 days a week for a total of four weeks. Average ± SD of the tumor volume for each group is shown; p-values were obtained using two-tailed t-test. *p < 0.05. H Kaplan–Meier curves relative to i.p. Leflunomide-treated AMO xenografts compared to control xenografts (log-rank-test, *p = 0.0094). Survival was evaluated from the first day of treatment until death or sacrifice. Percentage of mice alive is shown. I Western blot analysis of MFN2 protein levels in tumors retrieved from Leflunomide- or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein expression represents the mean ± SD from Leflunomide-or vehicle-treated tumor xenografts after normalization. **p < 0.01. L IHC analysis (20 × magnification) of Ki-67 expression in AMO xenografts retrieved from mice, four weeks after Leflunomide treatment; representative images are reported. M Western blot analysis of BAK, BIK, BIM, BID, NOXA and PUMA protein levels in tumors retrieved from Leflunomide-or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from Leflunomide-or vehicle-treated mice after normalization

Fig 3: Hypertension induced neurodegenerative alterations and mitochondrial injury. A Confocal images showing double immunofluorescence labeling for Nile-red (red) and Dapi (yellow) in the sections of prefrontal cortex (PFC) from sham- and DOCA-salt-treated mice. Scale bar, 10 μm. B Summary data for the Nile-red staining. Each datapoint represents a mouse. C and D Golgi staining for neuronal dendritic spines in the PFC sections from sham- and DOCA-salt-treated mice. C A representative image of Golgi-stained neurons and a respective trace image of dendritic branch structures. Scale bar, 20 μm. D Summary data for spine density in primary and secondary dendritic branches. The dendritic spine density was represented by the number of spines per 20 μm of dendritic length (n = 3 to 4 mice per group). E Representative TEM images for mitochondria in the PFC neurons of mice with sham- or DOCA-salt treatment. Arrows indicate neuronal mitochondria. Scale bar, 200 nm. Each datapoint corresponds to a sample. F Summarized percentages of damaged mitochondria (showing vacuoles) in each group. G Representative blots showing NADH dehydrogenase Fe-S protein 1 (NDUFS1) and cytochrome c (Cyto C) protein in cytoplasm (upper panel) and mitochondria (lower panel) fractions extracted from the PFC of mice. GAPDH and Cytochrome c oxidase (Cox) IV were used as loading controls. H Quantitative analysis for Cyto C protein expression in mitochondria (left) and NDUFS1 protein expression in cytoplasm (right). Data of each column was obtained from ≥ 3 individual experiments. I Representative blots for phospho-tau AT8 and Pan-lysine-succinylation (SL) in the PFC homogenates from mice after DOCA-salt or sham treatment. GAPDH was used as a loading control. Tau was used to obtain the ratio of tau-phosphorylation. J Summary of AT8 and SL60 (40~70 kDa proteins) expression levels based on densitometric analysis. Data of each column was obtained from >3 individual experiments. Data are shown as mean ± SEM. Statistical significance: Student’s t test: *P< 0.05, **P < 0.01, ****P < 0.0001 vs. sham

Fig 4: Promotion of mitochondrial fusion via genetic MFN2 upregulation replicates MARCH5 loss and triggers anti-myeloma responses. A Western blot analysis of NDUFS1, COX1, COX4, and MFN2 proteins in independent AMO-BZB cell clones (CL.15 and CL.19) with doxycycline-inducible MFN2 expression. GAPDH was used as a loading control. B Histogram bars reporting real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO-BZB cell clones (CL.19) with doxycycline-inducible MFN2 expression, 72 h after treatment. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. *p < 0.05. C) Cell viability was assessed by Cell Titer Glo assay in AMO-BZB cell clones (CL.15 and CL.19) with doxycycline-inducible MFN2 expression, 6 and 9 days after treatment with doxycycline. *p < 0.05; **p < 0.01. D Flow cytometry analysis of Annexin V/7AAD-stained AMO-BZB cell clone (CL.19) with doxycycline-inducible MFN2 expression, 6 and 9 days after treatment with Doxycycline. E Western blot analysis of PUMA and NOXA expression in AMO-BZB cell clones (CL.15; CL.19); GAPDH was used as loading control. F In vivo tumor growth evaluation of subcutaneous xenografts with AMO-BZB (CL.19), receiving doxycycline or vehicle as control; administrations were performed via drinking water. Average ± SD of the tumor volume for each group is shown; p-values were obtained using two-tailed t-test. **p < 0.001. G Kaplan–Meier curves relative to AMO-BZB xenografts (CL.19) receiving doxycycline or vehicle as control (log-rank-test). Survival was evaluated from the first day of treatment until death or sacrifice. Percentage of mice alive is shown. H Western blot analysis of MFN2 protein levels in tumors retrieved from doxycycline or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from doxycycline-or vehicle-treated mice after normalization. I IHC analysis (20 × magnification) of Ki-67 expression in xenografts from AMO-BZB cells (CL.19) with doxycycline-inducible MFN2, retrieved from mice before sacrifice; representative images are reported. L Western blot analysis of BAD, BID, NOXA and PUMA protein levels in retrieved xenografts; GAPDH was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from doxycycline-or vehicle-treated mice after normalization *p < 0.05; **p < 0.01

Fig 5: One-dimensional Western blot analysis with anti-PDHA1 (A), anti-NDUFS1 (B), anti-LDHA (C), and anti-LDHB (D) on soleus samples from WT and IT mice. Kruskal–Wallis statistical analysis with Dunn correction was performed. * indicate significant levels p ≤ 0.05.