Fig 1: Effect of the m.9396G > A variant on transcript and protein levels. (A) Relative MT-CO3 mRNAs levels in cybrids M1 and M2 relative to that in WT cybrids. (B) Relative MT-CO3 protein levels in cybrids WT, M1, and M2 determined by Western blotting. VDAC was used as the internal control. (C) For cybrids WT, M1, and M2, IGA analysis of CIV and BN-PAGE/immunoblot analysis of MRC complexes using antibodies against subunits of complexes I (NDUFS2), II (SDHB), III (UQCRC2), IV (COXIV), and V (ATPB). VDAC was used as the internal control. CI, complex I; CII, complex II; CIII, complex III; CV, complex V; SC, supercomplex.
Fig 2: A direct interaction between MCU and Complex I alters uniporter stability.a NDUFA13 co-immunoprecipitates with MCU-Flag. Images are representative of 3 separate trials. b Left, mVenus-tagged Complex I subunits surveyed for FRET with MCU-mCerulean via flow cytometry. Images above each graph show mVenus-tagged constructs and MitoTracker Orange (MTO). Within graphs, each point in the density plot is an individual cell expressing MCU-mCerulean and the corresponding mVenus-tagged construct. For each cell, the FRET efficiency between its mCerulean and mVenus is displayed in the y axis, while the degree of expression of the mVenus-tagged construct is revealed by the mVenus fluorescence in the x axis. Right, summary of FRET efficiency between MCU-mCerulean and the corresponding mVenus-tagged constructs at moderate mVenus expression. c Left, MCU-NDUFS2 Duolink colocalization occurs in mitochondria (CoxIV) and is more prevalent at baseline (WT) than after Complex I inhibition (Rotenone). Right, Duolink summary. Note that 74% of MCU-ATP5A and 85% of Rotenone-treated (MCU-S2) cells had zero Duolink spots, compared to 27% of MCU-NDUFS2 and 37% of MCU-MTCO1 cells. d Left, MCU-NDUFS2 Duolink greater in control than NDUFB10-/C107S IPSCs. Right, Summary. 45% of NDUFB10-/C107S IPSCs had zero Duolink spots, compared to 19% for control. Summary data are presented as mean values ± SEM. Statistics: (b, c) 1-way ANOVA followed by Bonferroni-corrected means comparisons; (d), two-sided Student’s t test. Source data are provided as a Source Data file.
Fig 3: Silencing of complex I subunits specifically affects the assembly of complex I-containing supercomplexes (a and b) Protein extracts from BAECs treated with siSCR, siNDUFS4 or siNDUFS2 were immunoblotted for NDUFS4 or NDUFS2 proteins with tubulin as loading control. Up: representative image; down: quantification of three independent experiments (mean±s.e.m.). (c and d) BN-PAGE of siSCR-treated or siNDUFS4-treated BAECs, analyzed by western blotting with antibodies against NDUFB6 (complex I; c) or Core I (complex III; d). Representative image of three independent experiments.
Fig 4: Interference or inhibition of complex I prevent the increase in ROS production triggered by hypoxia. (a-c) Detection of superoxide production by fluorescence microscopy in fixed cells. Cells were incubated for 60 min in normoxia (Nx), for 30 min in normoxia with antimycin A (AA 10 µM) or incubated with pre-hypoxic medium in a hypoxia chamber at 1% O2 (Hp) for 0, 15, 30, 45 or 60 min. DHE (5 µM) was added for additional 10 min and cells were fixed in the hypoxia chamber. (a and b) BAECs were treated with scramble siRNA (siSCR; black bars) or siRNA against NDUFS4 (a) or NDUFS2 (b). (c) BAECs were untreated (Control) or treated with 1 µM rotenone (Rot 1 µM). Data are presented as the mean±s.e.m. of three independent experiments. n.s. non-significant difference, *p<0.05, **p<0.01 and ***p<0.001 (ANOVA with Tukey post hoc test); only the significances between control normoxia and control hypoxia 0–10 min or treated hypoxia 0–10 min groups are shown. (d-f) Detection of ROS by the ratiometric fluorescent protein HyPer. (d) BAECs were transfected with CytoHyPer, treated with 2 mM of dithiothreitol (DTT) and with 30 µM antimycin A (AA). (e and f) CytoHyPer-transfected BAECs either untreated (e) or treated with 1 µM rotenone (f) were subjected to normoxia (Nx, •) or hypoxia (1% O2; Hp, ?). Data are presented as the mean±s.e.m. of four independent experiments. n.s. non-significant difference, *p<0.05 Hp vs. Nx (Mann-Whitney U test).
Fig 5: SMYD1a overexpression regulates mitochondrial respiration by increasing the formation of supercomplexes and cristae. A Diagram showing factors influencing mitochondrial respiration. B Western blotting evaluation of the electron transport chain (ETC) complex subunits using an antibody cocktail showing no significant changes in abundance of ETC complexes in TG mice or after PO, n = 3. C Immunoblotting for additional ETC subunits showed D modest changes in a few subunits (NDUFS2, NDUFV1), but no change in most of those examined, n = 3. E–F Mitochondrial biogenesis is absent in transgenic SMYD1a mice as confirmed by three markers: E mitochondrial DNA quantified by ND1 and 16S rRNA via qPCR, n = 6, and F citrate synthase activity in TG and WT mice which showed no change after 2 weeks of DOX-induced SMYD1a overexpression, n = 3–4. G–I SMYD1a overexpression increases supercomplex formation as determined by blue-native PAGE gel stained with G Coomassie, evaluated for H Complex I activity and I quantified in WT and TG mice 48 h after permanent occlusion of the LAD or Sham surgery. Asterisk * indicates p < 0.05, n = 3–4. J Electron micrographs of cardiac tissue from WT, SMYD1a transgenic mice and Smyd1 KO mice show that while loss of SMYD1 leads to loss of cristae structure, SMYD1a overexpression leads to K larger mitochondria with L more dense, narrower cristae. Asterisk * indicates p-value p < 0.05, n = 3–7. M,N) Western blotting and quantification analysis showing increased expression of OPA1 and MFN2 and no change in mitofillin expression in TG mice, as compared to WT controls. Asterisk * indicates p < 0.05, n = 3. O SMYD1a overexpression leads to the formation of ETC supercomplexes and more dense and narrower cristae that results in increased mitochondrial respiration and ATP production through which it ultimately reduces ischemic injury and pathological remodeling
Supplier Page from Abcam for Anti-NDUFS2 antibody [7A12BE5AD5]