Fig 1: Hypoxia promotes loss of mitochondrial genes and genes involved in energy-consuming processes.a Schematic diagram shows which datasets were compared in our analysis (hypoxia-glucose vs. normoxia-glucose). Sequencing reads from triplicate incubations were analysed by the MAGeCK analysis platform, and relative sgRNA abundances were calculated between experimental conditions. b Charts show FDR-corrected significance values of all sequenced genes, with significantly enriched (blue circles) or depleted (brown circles) sgRNAs in cells cultured in hypoxia-glucose compared to normoxia-glucose. n = 3. p < 0.05, FDR < 30%. c Pie charts show number of mitochondrial genes among the genes identified with significantly enriched or depleted sgRNAs from cells in (a). d Panel shows 31 selected mitochondrial genes with significantly enriched sgRNAs from (b). Genes significantly depleted in normoxia-glucose (plasmid vs. library) are highlighted in brown. e Western blots show expression of NDUFB10, SDHA, UQCRC2, COXIV, and BNIP3 in U2OS and HCT116 cells incubated for 5 days in normoxia (Nor) or hypoxia (Hyp, 1% O2). ß-Actin used as a loading control. f Chart shows overrepresentation analysis of all genes with significantly enriched sgRNAs in hypoxia (hypoxia-glucose vs. normoxia-glucose). Gene sets involving translation (mRNA splicing and processing of pre-mRNA), and regulation of actin cytoskeleton are highlighted (*). g, h Schematic diagram shows clusters of interacting genes with significantly enriched sgRNAs in hypoxia (hypoxia-glucose vs. normoxia-glucose) involved in mRNA processing (g), and cytoskeleton arrangement (h).
Fig 2: POLG mutations induced defects in respiratory chain complex I AComplex I immunohistochemistry in the occipital cortex of a neurologically healthy control (a) and two patients with POLG disease (b and c) (Scale bar, 20 µm). Patients have numerous complex I-negative neurons (examples marked by arrows). (d) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases (n = 5) and neurologically healthy controls (n = 5). Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons. For the purposes of comparison, a control sample has been arbitrarily set to one. The medians of the two groups are compared by Mann–Whitney U-test. Data are presented as mean (horizontal bars) ± SEM (vertical bars).BRepresentative confocal images of immunostaining for mitochondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A, and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).C–EFlow cytometric measurements of mitochondrial complex I (C, n = 4, technical replicates per clone), II (D, n = 3, technical replicates per clone for control; n = 4, technical replicates per clone for WS5A and CP2A) and IV (E, n = 3, technical replicates per clone for control and CP2A; n = 4, technical replicates per clone for WS5A) protein level in iPSC-derived NSCs. Expressed as specific complex I, II, and IV level (total complex I, II, IV level/TOMM20).F–HFlow cytometric measurements of mitochondrial complex I (F, n = 4, technical replicates per clone), II (G, n = 4, technical replicates per clone) and IV (H, n = 4, technical replicates per clone) protein level in iPSCs. Expressed as specific complex I, II, and IV level.I–KFlow cytometric measurements of mitochondrial complex I (I, n = 4, technical replicates), II (J, n = 4, technical replicates), and IV (K, n = 4, technical replicates) protein level in parental fibroblasts expressed as specific complex I, II, and IV level.Data information: The data points in C represent NSCs generated from 5 different controls, including 3 clones from Detroit 551 control, 1 clone from control AG05836, 1 clone from control CCD-1079Sk, 3 clones from WS5A patient iPSCs, and 2 clones from CP2A patient iPSCs. The data points in D, E represent NSCs generated from 3 clones from Detroit 551 control, 3 clones from WS5A patient iPSCs, and 2 clones from CP2A patient iPSCs. The data points in F–H represent 2 clones from Detroit 551 iPSCs, 3 clones from WS5A patient iPSCs, and 2 clones for CP2A iPSCs. Data are presented as mean ± SEM for the number of samples. Mann–Whitney U-test was used for the data presented in F and J. Two-sided Student's t-test was used for the data presented in C–E, G–I and K. Significance is denoted for P values of less than 0.05. *P < 0.05; ***P < 0.001; ****P < 0.0001.Source data are available online for this figure.
Fig 3: Summary diagram showing dynamics of mitochondria content and ROS generation during the course of reprogramming of MEFs to iPSCs. Inhibiting CI of ETC via Ndufs1/Ndufb10 knockdown or treatment with rotenone are well tolerated during the first 3 days, however, strongly suppresse cell reprogramming during intermediate and late stages of this process. ROS scavenging by antioxidants ameliorates cell reprograming during the first 3 days, however, it suppresses the process when applied at later stages.
Fig 4: Immunofluorescence staining for CI and aggregated a-synuclein in dopaminergic neurons of the SNc. The images show representative examples of fluorescence immunoreactivity against the mitochondrial outer membrane marker (VDAC1, red), CI (NDUFB10, green), aggregated a-syn (5G4, cyan), dsDNA (DAPI, blue), and a combination of all channels. CI-positive neurons were either 5G4-negative (A), or 5G4-positive with punctate inclusions (B) or LB/PB (C). CI-negative neurons were either 5G4-negative (D), or positive with punctate inclusions (E). Neuromelanin is marked with an asterisk. Scale bar: 10 microns. The plots in (F) depict the total percentage of neurons in each a-syn inclusion state (LB or PB/punctate inclusions/no inclusion) within each CI state (positive/negative) from each of the observers.
Fig 5: 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.
Supplier Page from Abcam for Anti-NDUFB10 antibody [EPR16230-47]