Fig 1: CLDN10 overexpression increases the levels of ROS, Cleaved-Caspase 3, E-cadherin and SDHB and decreases the levels of N-cadherin and mitochondrial membrane potential. A, Comparison of the ROS levels in CLDN10 overexpression and the control Caki-1 cells. B, Comparison of the expression of Cleaved-Caspase 3, E-cadherin and N-cadherin in CLDN10 overexpression and the control Caki-1 cells. C, Comparison of the expression of SDHB in CLDN10 overexpression and the control Caki-1 cells. D, Comparison of mitochondrial membrane potential in CLDN10 overexpression and the control Caki-1 cells (when the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria and forms JC-1 aggregates, which produces red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria, at this time, JC-1 is a monomer and can produce green fluorescence). E, Comparison of the ROS levels in ATP5O knockdown and its control Caki-1 cells. F, Comparison of the expression of Cleaved-Caspase 3, E-cadherin and N-cadherin in ATP5O knockdown and its control Caki-1 cells. G, Comparison of the expression of SDHB in ATP5O knockdown and its control Caki-1 cells. H, Comparison of mitochondrial membrane potential in ATP5O knockdown and its control Caki-1 cells. I, The effects of NAD+ inhibiting ATP5O acetylation on the expression of ATP5O, NDUFS2, Cleaved-Caspase 3, N-cadherin, E-cadherin and SDHB. ***P < 0.001, ****P < 0.0001.
Fig 2: CLDN10 overexpression increases NDUFS2 expression by regulating ATP5O. A, The expression of ATP5O was knocked down in CLDN10 overexpression Caki-1 cells. B, Comparison of the differential protein between ATP5O knockdown and its control Caki-1 cells by TMT experiment. C, Venn diagram analysis of the two TMT test data. D, The correlation between ATP5O and NDUFS2 expression, and CLDN10 and DNUFS2 expression were analyzed using TCGA-KIRC data (n=539). E, Comparison of the expression of NDUFS2 in the total protein and mitochondrial protein of CLDN10 overexpression and the control Caki-1 cells. F, Comparison of the expression of NDUFS2 in the total protein and mitochondrial protein of ATP5O knockdown and its control Caki-1 cells. G, The comparison of NDUFS2 expression in ccRCC (n=539) and adjacent normal renal tissues (AN, n=72), T1/T2 group (n=340) and T3/T4 group (n=190), G1/G2 group (n=241) and G3/G4 group (n=281) and Stage I/II group (n=322) and Stage III/IV group (n=205) and the relationship between NDUFS2 expression and the prognosis of ccRCC patients were analyzed using TCGA-KIRC data.
Fig 3: Rescue of the secretory response to hypoxia of mitochondrial complex I-deficient glomus cells by conditional transgenic NDI1 expression in adulthood.a Histological sections of the carotid body (CB) from ESR-WT (top) and ESR-KO/NDI1 (bottom) mice illustrating immunoreactivity for GFP (green, indicating NDI1 expression) and TH (red). Similar studies were performed in 4 mice for each genotype. DAPI was used to stain nuclei (blue). Calibration bar (10 µM) applies to all panels. b Histological sections illustrating TH (green) and NDUFS2 (red) protein expression in CB glomus cells from ESR-WT (top), ESR-KO (middle) and ESR-KO/NDI1 (bottom) mice. Nuclei were stained with DAPI (blue). Similar immunocytochemical studies were performed in n = 4 mice for each genotype. Calibration bar (10 µM) applies to all panels. c–e Representative amperometric recordings of the secretory activity induced by hypoxia (O2 tension ~15% mmHg) hypercapnia (20% CO2) and depolarization with potassium (40 mM K) in carotid body glomus cells from ESR-WT (c), ESR-KO (d) and ESR-KO/NDI1 (e) mice. Calibration bars as indicated in (c). f–h Representative recordings of the secretory activity induced by rotenone (5 µM) and hypoxia in the presence of rotenone in glomus cells from ESR-WT (f), ESR-KO (g) and ESR-KO/NDI1 (h) mice. Calibration bars are indicated in (c). i–k Basal secretion rate (picoCoulombs/min) in normoxia and the secretory response to hypoxia and hypercapnia (CO2) of glomus cells in CB slices from the mouse models studied. In all cases n = cells/mice. Normoxia (i): ESR-WT, 0.17 ± 0.04, n = 11/9; ESR-KO, 0.06 ± 0.02, n = 8/4; ESR-KO/NDI, 0.11 ± 0.03, n = 11/5. Hypoxia (j): ESR-WT, 2.90 ± 0.35, n = 11/9; ESR-KO, 0.25 ± 0.11, n = 8/4; ESR-KO/NDI, 3.14 ± 0.77, n = 11/5. CO2, (k): ESR-WT, 3.31 ± 1.40, n = 8/7; ESR-KO, 2.37 ± 0.42, n = 8/4; ESR-KO/NDI, 1.54 ± 0.37, n = 6/4. Data are expressed as mean ± SEM with all data values superimposed. Statistically significant P values, calculated by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, are represented in each panel. l, m Average secretion rate (picoCoulombs/min) induced by rotenone (l) and hypoxia in the presence of rotenone (m) in glomus cells in CB slices from the mouse models studied. In all cases n = cells/mice. ESR-WT (rotenone: 1.89 ± 0.28, n = 4/3; rotenone + hypoxia: 0.56 ± 0.16, n = 4/3); ESR-KO (rotenone: 0.08 ± 0.04, n = 5/2; rotenone + hypoxia: 0.26 ± 0.17 n = 5/2) and ESR-KO/NDI1 (rotenone: 0.21 ± 0.08, n = 6/4; rotenone + hypoxia: 2.71 ± 0.61 n = 5/3). In (l, m) P values represented in each panel were calculated by one way ANOVA followed by Tukey’s multiple comparisons post hoc test. Source data are provided as a Source Data file.
Fig 4: NDI1 expression and mitochondrial bioenergetic restoration in complex I-deficient carotid body glomus cells.a Histological sections of the carotid body (CB) from WT (left) and KO/NDI1 (right) mice illustrating colocalization of NDI1 expression (green fluorescent protein, GFP) and tyrosine hydroxylase (TH). DAPI was used to stain nuclei. Similar immunocytochemical studies were performed in n = 4 mice for each genotype. Calibration bar (10 µM) applies to all panels. b NDI1 mRNA levels, relative to WT, in CB samples from WT mice (blue dots, 0.030 ± 0.0009, n = 4 replicates/group), KO mice (yellow dots, 0 ± 0, n = 4 replicates/group) and KO/NDI1 mice (brown dots, 0.56 ± 0.14, n = 4 replicates/group). Data are expressed as mean ± SEM. P values calculated by one-way ANOVA followed by Newman-Keuls multiple comparisons test are indicated. c Immunostaining of CB sections illustrating NDUFS2 protein expression in TH positive cells from WT mice (left) and the disappearance in CB cells from KO (middle) and KO/NDI1 (right) mice. Similar immunocytochemical studies were performed in n = 4 mice for each genotype. Calibration bar (10 µM) applies to all panels. d Ndufs2 mRNA levels, relative to WT, in CB samples from WT mice (blue dots, 1 ± 0.3, n = 4 replicates/group), KO mice (yellow dots, 0.1 ± 0.04, n = 4 replicates/group) and KO/NDI1 mice (brown dots, 0.24 ± 0.07, n = 3 replicates/group). Data are expressed as mean ± SEM. P values calculated by one-way ANOVA followed by Newman-Keuls multiple comparisons test are indicated. e Representative time-lapse measurements of the ATP/ADP ratio estimated in glomus cells in CB slices expressing PercevalHR obtained from the various mouse models studied. Oligomycin (10 µM) and 2-deoxyglucose (5 mM glucose in the external solution was replaced with 5 mM 2-DG) were applied to determine the contribution of oxidative phosphorylation (OXPHOS) and glycolysis to the ATP/ADP ratio. Recordings are normalized to the value at the onset of each experiment. f Distribution of the OXPHOS index (OXPHOS/OXPHOS + glycolysis) in glomus cells from the mouse models studied. WT (blue, n = 8/5 cells/mice), KO (yellow, n = 8/4 cells/mice) and KO/NDI1 (brown, n = 9/4 cells/mice). The boxplots represent median (middle line), 25th, 75th percentile (box), and largest and smallest values range (whiskers). Statistically significant P values, calculated by non-parametric Kruskal-Wallis tests followed by Dunn’s post hoc test. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-NDUFS2 antibody [EPR16266]