Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 1. Mitochondria-nucleus contacts. Mesenchymal stem cells (MSCs) were cultured in low-glucose (LG), adipogenic (AD) and osteogenic (OS) media for 3, 7, and 21 days, as indicated. (a) Representative 3D images of mitochondrial morphology, as detected by anti-TOM20 Ab immunofluorescence, and contact sites identified by creating the isosurface of the colocalization channel between nuclear staining (Hoechst) and TOM20. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). (b) Quantification of mitochondrial total volume, number, and total volume and number of contacts. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments. Data are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Staining, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 2. Mitochondria in MSC differentiation processes. (a) Representative immunoblot and (b) quantification of HSP60, TOM20, VDAC, and TIM23 protein levels normalized to GAPDH levels. (c) XF phenograms representing the metabolic switching of MSCs during differentiation, as detected using an Extracellular Flux Analyzer (Seahorse Bioscience). (d,e) Oxygen consumption rate (OCR) measurements and relative derived parameters after the addition of oligomycin [1 µM], 1 µM carbonylcyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP) [1 µM], and antimycin A [2 µM] + rotenone [2 µM] in MSCs cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media for (d) 7 and (e) 21 days. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Derivative Assay, Cell Culture

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 4. Inhibition of the citrate transport system impaired mitochondrial behavior. MSCs were cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media; where indicated, BTC [5 mM] and iCTP [500 µM] were added to inhibit the transport of citrate produced in mitochondria to the cytosol. (a) Representative images and (b) analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Inhibition, Cell Culture, Produced, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 5. Citrate from mitochondria increases the level of α-ketoglutarate to promote osteogenesis. (a) Citrate can be converted to acetyl-CoA by citrate lyase (inhibited by BMS) or to α-ketoglutarate (αKG), two metabolites that can mediate mitochondrion-nucleus communication. (b,c) Analysis of relative mRNA levels of osteogenic markers (RUNX2, RANKL, osteocalcin (OC), osteopontin (OPN), osterix (OSX), and alkaline phosphatase (ALP)) in MSCs cultured in low-glucose (LG) and osteogenic media (OS); where indicated, BMS [1 mM], BTC [5 mM], and αKG [1 mM] were added for 21 days. (d) Representative images and analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites in MSCs cultured in LG and osteogenic media (OS); where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. (e) Representative images and quantification of H3K9me3 in MSCs cultured in LG and OS media; where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. Magnification 40x. Scale bar 10 µm (in zoom 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Derivative Assay

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: AS-IV improved mitophagy and attenuated mitochondrial membrane potential loss in BLM-induced VSMCs. A After administration of AS-IV, MMP was identified by TMRM staining in BLM-induced VSMCs. Magnification, 200 × , scale bar = 50 μm. B TEM was used to observe the influence of AS-IV on the mitochondrial structure of BLM-induced VSMCs. C Flow Cytometer was used to assess the impact of AS-IV on the TMRM + fluorescent intensity in BLM-stimulated VSMCs. D P62 expression was examined by Western blot in BLM-induced VSMCs, which were given AS-IV, and the P62/TOMM20 value was determined. E expression of Parkin was measured by Western blot in BLM-induced VSMCs. LD low dose, MD medium dose, HD high dose. * P < 0.05, ** P < 0.05, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Membrane, Staining, Flow Cytometry, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: Interleukins-27 Aggravates Liver Injury by Impairing the Antimicrobial Response of Macrophages via the Promotion of Mitochondrial Dysfunction in the Context of Sepsis

doi: 10.1155/mi/6608718

Figure Lengend Snippet: IL-27 increased LPS-induced mitophagy in macrophages. (A) The number of mitochondria was detected using MitoTracker ( n = 4). (B) MitoTracker red fluorescence quantification analysis ( n = 4; Control vs. LPS p < 0.0001, LPS vs. LPS + IL-27 p =0.0004). (C) Western blotting was used to detect the mitophagy-related protein expression ( p -Parkin, Parkin, PINK1, and LC3) ( n = 4). (D–G) Relative statistical analysis of p -Parkin/Parkin ( n = 4; Control vs. LPS p < 0.0001, LPS vs. LPS + IL-27 p =0.1192), PINK1 ( n = 4; Control vs. LPS p =0.0001, LPS vs. LPS + IL-27 p =0.0002), LC3 I ( n = 4; Control vs. LPS p =0.0057, LPS vs. LPS + IL-27 p =0.0649) and LC3 II ( n = 4; Control vs. LPS p =0.0038, LPS vs. LPS + IL-27 p < 0.0001) protein expression. (H) TOM20 and LC3 immunofluorescence staining were used to assess mitophagy ( n = 4). (I) Western blotting was used to detect the hepatic p -Parkin/Parkin, PINK1, LC3 I, and LC3 II ( n = 3) protein expression. (J–M) Statistical analysis of relative hepatic p -Parkin/Parkin ( n = 3; WT vs. CLP p =0.0018, CLP vs. IL-27R −/− +CLP p =0.0065), PINK1 ( n = 3; WT vs. CLP p < 0.0001, CLP vs. IL-27R −/− +CLP p =0.0003), LC3 I ( n = 3; WT vs. CLP p < 0.0001, CLP vs. IL-27R −/− +CLP p < 0.0001) and LC3 II ( n = 3; WT vs. CLP p =0.0154, CLP vs. IL-27R −/− +CLP p =0.0077) protein expression. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001, ⁣ ∗∗∗∗ p < 0.0001. CLP, cecal ligation and puncture; IL, interleukin; LPS, lipopolysaccharide; WT, wild-type.

Article Snippet: Following the block with goat serum at room temperature for 45 min, the cells were incubated overnight at 4°C with primary antibodies targeting TOM20 and LC3 and then with FITC- or CY3-conjugated secondary antibodies at room temperature for 30 min (1:200, Affinity Biosciences).

Techniques: Fluorescence, Control, Western Blot, Expressing, Immunofluorescence, Staining, Ligation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: M1 Microglia Induced Neuronal Injury on Ischemic Stroke via Mitochondrial Crosstalk between Microglia and Neurons

doi: 10.1155/2022/4335272

Figure Lengend Snippet: Mitochondrial changes in activated microglia (M1). (a) Schematic diagram of intracellular mitochondrial fusion and fission process in microglia after OGD/R. (b) Western blot assay of mitochondrial fusion protein (Opa1 and Mfn1), TOM20, and cytochrome c in mitochondria of M0 and M1 microglia ( n = 3). Results are displayed in a form of mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (c) Western blotting findings of mitochondrial fission protein including MFF, Fis1, Mid49, and Mid51 in mitochondria of M0 and M1 microglia ( n = 3). Data presented are mean ± SD; ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (d) Mitochondrial function in M1 microglia and M0 microglia was investigated by determining ATP ( n = 3), mitochondria membrane potential ( n = 3), and ROS ( n = 3). The relative ratio of intracellular ATP was determined by calculating the ratio of level of ATP in M0-BV2 and M1-BV2 cells to level of ATP in M0-BV2 cells. Results are displayed in a form of mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (e) Morphology of intracellular mitochondria in BV2 cells visualized under TEM, scale bar: 1.0 μ m.

Article Snippet: Rabbit antibodies, such as TOM20 (WL0706), BAX (WL01637), BCL-2 (WL01556), cytochrome c (WL02410), caspase 3 (WL04004), iNOS (WL0992a), VDAC (WL02790), IL-2 (WL03259), TNF- α (WL01581), IL-1 β (WLH3903), and β -actin (WL01372), were procured from Wanleibio in Shenyang, China, while rabbit anti-MFF (AF2365), anti-Mid49 (DF12044), anti-Mid51 (DF12019), anti-Opa1 (DF8587), and anti-Mfn1 (DF7543) were obtained from AFFINITY, Co., Ltd.

Techniques: Western Blot, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: M1 Microglia Induced Neuronal Injury on Ischemic Stroke via Mitochondrial Crosstalk between Microglia and Neurons

doi: 10.1155/2022/4335272

Figure Lengend Snippet: Functional changes of released mitochondria secreted by activated microglia (M1). (a) Morphological characterization of released mitochondria isolated from culture medium of M0 microglia (Mito/M0-BV2) and M1 microglia (Mito/M1-BV2). Scale bar: 1.0 μ m. (b) Levels of proteins associated with mitochondrial fission in Mito/M0-BV2 and Mito/M1-BV2 based on the western blotting ( n = 3). Results are displayed in a form of mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (c) The expression of associated fusion protein, TOM20, and Cyto C in Mito/M0-BV2 and Mito/M1-BV2 ( n = 3). Results are displayed in a form of mean ± SD, ∗ P < 0.05, and ∗∗∗ P < 0.001. (d) After OGD/R for 24 hours, function of Mito/M0-BV2 and Mito/M1-BV2 was detected by checking ATP ( n = 3), mitochondria membrane potential ( n = 3), and ROS ( n = 3). The relative ratio of extracellular ATP in secreted mitochondria from BV2 cells was determined by calculating the ratio of level of ATP in Mito/M0-BV2 and Mito/M1-BV2 to level of ATP in Mito/M0-BV2. Results are displayed in a form of mean ± SD; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Rabbit antibodies, such as TOM20 (WL0706), BAX (WL01637), BCL-2 (WL01556), cytochrome c (WL02410), caspase 3 (WL04004), iNOS (WL0992a), VDAC (WL02790), IL-2 (WL03259), TNF- α (WL01581), IL-1 β (WLH3903), and β -actin (WL01372), were procured from Wanleibio in Shenyang, China, while rabbit anti-MFF (AF2365), anti-Mid49 (DF12044), anti-Mid51 (DF12019), anti-Opa1 (DF8587), and anti-Mfn1 (DF7543) were obtained from AFFINITY, Co., Ltd.

Techniques: Functional Assay, Isolation, Western Blot, Expressing, Membrane