Fig 1: OXPHOS levels in hypothalamic mitochondria of 3xTg male and female mice compared to controls. Western blot images show representative bands for NADH dehydrogenase beta subcomplex subunit 8 of Complex I (NDUFB8; A), succinate dehydrogenase subunit B of complex II (SDHB; B), cytochrome b-c1 complex subunit 2 of complex III (UQCRC2; C), cytochrome c oxidase subunit 1 of complex IV (MTCO1; D), and ATP synthase subunit alpha of complex V (ATP5A; E). Quantification of these proteins, normalized to total protein (F) for 3xTg female and male mice and their respective controls at 2, 6, and 13 months of age. Asterisks indicate statistical significance compared to corresponding control groups. Results are presented as mean ± SD of n = 4 per group, with significance levels denoted as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, analyzed using functional three-way ANOVA. In female 3xTg mice, complex II expression was significantly reduced compared to males, while no sex-based differences were observed in control mice across all age groups (A). Complex III levels were higher in female 3xTg mice at 2 and 6 months, whereas control mice showed an increase only at 13 months (B). No significant differences in complex V expression were noted in 3xTg mice; however, at 2 months, control mice exhibited higher levels in females, with no notable differences in other age groups (C). Additionally, there were no significant variations in the levels of protein subunits for complexes I and IV across all age groups, regardless of genotype or sex (D)
Fig 2: Mitochondrial proteins are differentially acetylated, and mitochondrial enzyme activity is compromised in Kat2 DKO.(A) Schematic of the pipeline for acetylomics MS. Created with Biorender.com. (B) GO term analysis of the proteome (top) and nonhistone proteins with down-regulated acetyl-lysine residues (bottom) from Kat2 DKO IECs (n = 4 controls and 6 DKOs). (C) Volcano plot of acetyl-lysine residues from acetylomics of Kat2 DKO IECs. Orange points indicate lysine residues on mitochondrial proteins (n = 4 controls and 6 DKOs). Stains for activity of mitochondrial enzymes (D) NADH and (E) COX in jejunal intestine sections (n = 2 to 4 per group). Scale bars, 500 μm. (F) COX activity in the IECs from control and Kat2 DKO mice (n = 3 mice, Welch’s t test) using spectrophotometry. ***P < 0.001.
Fig 3: Treatment with aporphine and isoquinoline derivatives induce an intrinsic apoptosis cell death. (A) COX activities of GBM cells after 24 h of incubation with A5 (30 μM) or C1 (30 μM). Results are expressed as percentage of cell control treated with DMSO. (B) Representative immunoblot of cleaved caspase-3 in U3017MG cells treated with TMZ, APO, A5 or C1 for 48 h. β-Actin was used as a loading control, and molecular mass (kDa) markers are indicated along with densitometric values of normalized band intensity. (C, D) RT-qPCR was used for detection of markers for cell death mediated by apoptosis: BIM, BECN1 and BAX in U3017MG (C) and U3031MG (D) cells treated with DMSO, TMZ (150 µM), APO (25 µM), A5 (30 µM) or C1 (30 µM) for 48 h. TMZ, an alkylating agent frequently used as chemotherapy in GBM patients, was used as the positive control. (E, F) RT-qPCR was used for detection of DNA damage response gene expression: CDKN1A (p21) in U3017MG (E) and U3031MG (F) cells treated with DMSO, TMZ (150 µM), APO (25 µM), A5 (30 µM) or C1 (30 µM) for 48 h. All the relative expression levels were normalized to GAPDH expression and calculated using the 2−ΔΔCt method. Error bars represent SD from three biological replicates and p values were calculated according to the two-way ANOVA test followed by Bonferroni (post-test); *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: xtCYTC transcripts, xtCYTC protein, and xtCOXIV enzymatic activity expressions under various environments. (a) Tissue distribution of xtCYTC transcripts in various tissues collected from habitat xtcrabs and aquarium xtcrabs (n = 6 crabs/group). aGi, anterior gill; pGi, posterior gill; St, stomach; DG, digestive gland; In, intestine; Mu, muscle; He, heart. (b) xtCYTC transcripts in pGi of habitat xtcrabs and aquarium xtcrabs (n = 36 crabs/group). (c) xtCYTC protein expression as measured by Western blot (with xtCYTC antiserum) in the pGi of habitat and aquarium xtcrabs. Selected blot images are depicted above the graphs. Actin was used as a reference protein. Quantification of signal intensity was performed with ImageJ software (n = 3 xtcrabs/group). (d) COXIV enzymatic activity in habitat xtcrabs and aquarium xtcrabs (n = 6 crabs/group). Different letters indicate one-way ANOVA in xtCYTC tissue distribution (p < 0.05). EF1A was used as an internal reference to normalize the gene expression levels. The highest value among groups is set at 100, and each value is normalized to the ratio (each value/highest value) x100. Asterisks indicate significant difference between groups by t-test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). The bars represent mean ± SD (standar deviation).
Fig 5: Reactivation of OxPhos attenuates ETP-ALL progression(A) Bar graph showing quantitative analysis of mean fluorescence intensity (MFI) of ROS shown in ETP-ALL cells treated with DCA (20 mM) at different time points.(B) Bar chart showing OCR in ZYXY-T1 and PEER cells upon DCA treatment (20 mM, 12 h).(C) Bar chart displaying mitochondrial ATP production in ZYXY-T1 and PEER cells treated with DCA (20 mM, 12 h).(D) Bar chart showing the apoptosis ratio (Annexin V/DAPI) in ETP-ALL (ZYXY-T1 and PEER) and T-ALL (Molt4 and Jurkat) cells following DCA treatment (0/6.25/12.5/25 mM; 24 h).(E) IC50 graph with a curve fit line showing the dose-dependent effects of DCA in ETP-ALL (ZYXY-T1 and PEER) and T-ALL (Molt4 and Jurkat) cells (0/3.125/6.25/12.5/25/50/100 mM; 24 h).(F) MTT assay demonstrating the cell viability of different primary cells following DCA treatment (20 mM; 24 h) in ETP-ALL primary cells.(G) Bar chart showing the apoptosis ratio (Annexin V/DAPI) in PEER cells after cotreatment with DCA (25 mM) and cytarabine (100 nM) or vincristine (5 nM) for 24 h.(H) Schematic representation of the experimental design and treatment (vehicle or DCA) for in vivo studies in NCG mice xenografted with ZYXY-T1 (n = 4 per group; 150 mg/kg/mouse; intraperitoneal injection and every other day, 7 times totally).(I and J) Violin plots showing the percentage of human ETP-ALL cells (human CD45+) engrafted in bone marrow (I) and spleen (J) of recipient NCG mice (n = 4 per group).(K) Violin plots showing the percentage of spleen weight vs. body weight.(L) Kaplan-Meier survival curve of NCG mice transplanted with ETP-ALL cells treated with vehicle or DCA (n = 5 per group).(M) Schematic representation of the experimental design and treatment (vehicle or DCA) for in vivo studies in NCG mice xenografted with primary ETP-ALL patient samples ETP-ALL-31 (n = 6 per group, 150 mg/kg/mouse; intraperitoneal injection and every other day, 12 times totally).(N) Representative flow cytometry dot plots illustrating bone marrow engraftment of human ETP-ALL cells in NCG mice.(O–Q) Violin plots showing the percentage of human ETP-ALL cells (human CD45+) engrafted in bone marrow (O), spleen (P), and peripheral blood (PB, Q) of recipient NCG mice (n = 6 per group).(R) Violin plot showing the weight of spleen.(S) Representative spleen of mice treated with vehicle or DCA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by one-way ANOVA (Figures 4A and 4D), two-way ANOVA (Figure 4B), unpaired t test (Figures 4C, 4F, 4G, 4J, 4K, 4P, 4Q, and 4R), or Log rank (Mantel-Cox) test (Figure 4L). Data were presented as the means ± SEM.
Supplier Page from Abcam for Cytochrome C Oxidase Assay Kit