Journal: Journal of Alzheimer's Disease

Article Title: Mitochondria-Targeted Antioxidants Protect Against Amyloid-β Toxicity in Alzheimer's Disease Neurons

doi: 10.3233/jad-2010-100564

Figure Lengend Snippet: Fig. 1. Representative immunoblots of peroxiredoxins, mitochondrial proteins, and neuroprotective proteins in control N2a neurons, Aβ-incubated N2a cells, and N2a neurons treated with MitoQ and SS31 and then incubated with Aβ (n = 3). A) Fifty µg of protein lysate was used from each cell sample; immunoblotting analysis was performed using antibodies of peroxiredoxins (1, 2, 3, 4, and 6). B) Drp1, Fis1, Mfn1, Mfn2, and Opa1. C) PGC1α, FOXO1, and NMDAR. Bottom panel shows immunoblotting of β-actin for equal loading. Lane 1 represents control N2a cells; lane 2, Aβ-incubated N2a cells; lane 3, resveratrol-treated N2a neurons; lane 4, N2a cells treated with resveratrol and then incubated with Aβ; lane 5, MitoQ-treated N2a cells; lane 6, N2a cells treated with MitoQ and then incubated with Aβ; lane 7, SS31-treated N2a cells; and lane 8, N2a cells treated with SS31 and then incubated with Aβ.

Article Snippet: All neurons were incubated overnight with a rabbit polyclonal anti-Drp1 primary antibody (Novus Biologicals) at a concentration of 1:400, at 4◦C.

Techniques: Western Blot, Control, Incubation

Journal: Journal of Alzheimer's Disease

Article Title: Mitochondria-Targeted Antioxidants Protect Against Amyloid-β Toxicity in Alzheimer's Disease Neurons

doi: 10.3233/jad-2010-100564

Figure Lengend Snippet: Fig. 6. Neurite outgrowth in control N2a cells, Aβ-incubated N2a cells, N2a cells treated with MitoQ and SS31 and then incubated with Aβ (n = 4). A presents representative images of (a) Drp1 immunostaining of control N2a cells, (b) Aβ-incubated N2a cells, (c) N2a cells treated with MitoQ and then incubated with Aβ, and (d) N2a cells treated with SS31 and then incubated with Aβ. White arrows indicate the loss of neuronal processes in the Aβ-incubated N2a cells, and red arrows indicate an increase in neuronal processes in N2a cells treated with MitoQ and SS31. B shows quantitative analysis of Drp1 immunostaining in N2a cells. (a) Significantly decreased Drp1 immunostaining in neuronal processes was found in Aβ-incubated N2a cells (P < 0.01) compared to control N2a cells, and significantly increased Drp1 immunostaining (neurite outgrowth) was found in neurons treated with MitoQ (P < 0.01) and SS31 (P < 0.03) compared to control N2a cells. (b) Significantly increased Drp1 immunostaining (neurite outgrowth) was found in N2a cells treated with MitoQ and SS31 and then incubated with Aβ (P < 0.01 and P < 0.03 respectively), compared to Aβ-incubated N2a cells.

Article Snippet: All neurons were incubated overnight with a rabbit polyclonal anti-Drp1 primary antibody (Novus Biologicals) at a concentration of 1:400, at 4◦C.

Techniques: Control, Incubation, Immunostaining

Journal: Journal of Alzheimer's Disease

Article Title: Mitochondria-Targeted Antioxidants Protect Against Amyloid-β Toxicity in Alzheimer's Disease Neurons

doi: 10.3233/jad-2010-100564

Figure Lengend Snippet: Fig. 9. Neurite outgrowth in primary hippocampal neurons from AβPP transgenic mice and wild-type mice with its hippocampal neurons treated with MitoQ, SS31, and resveratrol. A shows representative Drp1-stained hippocampal neurons: (a) wild-type, (b) AβPP transgenic, (c) MitoQ-treated AβPP, (d) SS31-treated AβPP, and (e) resveratrol-treated AβPP transgenic. Immunostaining was conducted using a Drp1 antibody (a marker for synaptic branching). White arrows indicate increased synaptic branching in hippocampal neurons treated with MitoQ and SS31. B) Cyclophilin D levels in hippocampal neurons from AβPP transgenic mice and wild-type mice. Image B shows representative CypD-stained hippocampal neurons: (a) wild-type, (b) AβPP transgenic, (c) MitoQ-treated AβPP transgenic, (d) SS31-treated AβPP transgenic, and (e) resveratrol-treated AβPP transgenic. Immunostaining was performed using the CypD antibody. C shows quantitative analysis of Drp1 immunostaining in hippocampal neurons from AβPP and wild-type mice. (a) Significantly increased Drp1 was found in hippocampal neurons from AβPP mice treated with MitoQ (P < 0.024) and with SS31 (P < 0.044), compared to control hippocampal neurons from AβPP transgenic mice. (b) Significantly increased Drp1 was found in hippocampal neurons from wild-type mice treated with SS31 (P < 0.03) and resveratrol (P < 0.038), compared to control hippocampal neurons from wild-type mice. D presents quantitative analysis of CypD in hippocampal neurons from AβPP transgenic mice and wild-type mice. (a) Significantly decreased CypD was found in hippocampal neurons from AβPP transgenic mice treated with SS31 (P < 0.0004), MitoQ (P < 0.006), and resveratrol (P < 0.013), compared to control hippocampal neurons from AβPP transgenic mice, and (b) hippocampal neurons from wild-type mice treated with SS31 and resveratrol showed significantly decreased levels of CypD, compared to control hippocampal neurons.

Article Snippet: All neurons were incubated overnight with a rabbit polyclonal anti-Drp1 primary antibody (Novus Biologicals) at a concentration of 1:400, at 4◦C.

Techniques: Transgenic Assay, Staining, Immunostaining, Marker, Control

Journal: Journal of Alzheimer's Disease

Article Title: Mitochondria-Targeted Antioxidants Protect Against Amyloid-β Toxicity in Alzheimer's Disease Neurons

doi: 10.3233/jad-2010-100564

Figure Lengend Snippet: Fig. 10. Double-labeling analysis of (a) Drp1 (synaptic branching protein), (b) CypD (mitochondrial matrix protein), and (c) the proteins Drp1 and CypD merged in MitoQ-treated AβPP transgenic mice hippocampal neurons.

Article Snippet: All neurons were incubated overnight with a rabbit polyclonal anti-Drp1 primary antibody (Novus Biologicals) at a concentration of 1:400, at 4◦C.

Techniques: Labeling, Transgenic Assay

Journal: Journal of translational medicine

Article Title: Pyruvate kinase M2 modulates mitochondrial dynamics and EMT in alveolar epithelial cells during sepsis-associated pulmonary fibrosis.

doi: 10.1186/s12967-025-06199-7

Figure Lengend Snippet: Fig. 2 LPS-induced PKM2 upregulation disrupts the imbalance of mitochondrial dynamics in sepsis-associated pulmonary fibrosis. (A) GO enrichment analysis of the control and LPS groups. (B) Mitochondrial morphology analysis of lung tissue by transmission electron microscopy. Scale bars = 20 μm (original magnification 3000×) and 5 μm (original magnification 8000×). (C) Protein expression levels of PKM2, DRP1 and MFN2 in the lung tissue of each group. (D) Immunofluorescence of PKM2, DRP1, MFN2 and E-cadherin in the lung tissue of each group. Scale bar = 30 μm (original magnification 600×). The values are presented as the means ± standard deviations, ***P < 0.001, ****P < 0.0001 vs. control group. LPS: lipopolysaccharide; PKM2: pyruvate kinase M2; DRP1: dynamin-related protein-1; MFN2: mitofusin-2

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany) for detection with specific primary antibodies including antibodies against collagen I (Col1a1, ab270993, Abcam), E-cadherin (14472, CST), alpha smooth muscle actin (α-SMA, A1011, ABclonal), vimentin (A19607, ABclonal), PKM2 (60268- 1-Ig, Proteintech), DRP1 (NB110-55288, Novus), and MFN2(9482 S, CST), followed by incubation with goat anti-rabbit (A0208, Beyotime) and anti-mouse (A0216, Beyotime) secondary antibodies.

Techniques: Control, Transmission Assay, Electron Microscopy, Expressing, Immunofluorescence

Journal: Journal of translational medicine

Article Title: Pyruvate kinase M2 modulates mitochondrial dynamics and EMT in alveolar epithelial cells during sepsis-associated pulmonary fibrosis.

doi: 10.1186/s12967-025-06199-7

Figure Lengend Snippet: Fig. 3 LPS-induced alveolar epithelial cell mesenchymal transition is correlated with PKM2 upregulation and imbalanced mitochondrial dynamics. MLE12 cells were treated with LPS for 24 h. (A) Protein expression levels of Col1a1, E-cadherin, DRP1, MFN2, PKM2, vimentin and α-SMA. (B) Immunofluorescence analysis of PKM2, DRP1 and MFN2 in MLE12 cells. Scale bar = 30 μm (original magnification 600×) (C) Mitochondrial morphology analysis of MLE12 cells by transmission electron microscopy. Scale bars = 20 μm (original magnification 3000×) and 5 μm (original magnification 8000×). The values are presented as the means ± standard deviations, ***P < 0.001, ****P < 0.0001 vs. control group. LPS: lipopolysaccharide; PKM2: pyruvate kinase M2; DRP1: dynamin-related protein-1; MFN2: mitofusin-2

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany) for detection with specific primary antibodies including antibodies against collagen I (Col1a1, ab270993, Abcam), E-cadherin (14472, CST), alpha smooth muscle actin (α-SMA, A1011, ABclonal), vimentin (A19607, ABclonal), PKM2 (60268- 1-Ig, Proteintech), DRP1 (NB110-55288, Novus), and MFN2(9482 S, CST), followed by incubation with goat anti-rabbit (A0208, Beyotime) and anti-mouse (A0216, Beyotime) secondary antibodies.

Techniques: Expressing, Immunofluorescence, Transmission Assay, Electron Microscopy, Control

Journal: Journal of translational medicine

Article Title: Pyruvate kinase M2 modulates mitochondrial dynamics and EMT in alveolar epithelial cells during sepsis-associated pulmonary fibrosis.

doi: 10.1186/s12967-025-06199-7

Figure Lengend Snippet: Fig. 4 Inhibition of PKM2 maintains mitochondrial dynamics and reduces alveolar epithelial cell mesenchymal transition. Shikonin was used to pretreat MLE12 cells at a concentration of 5 µmol/L, 12 h prior to LPS treatment in the in vitro model. (A) Protein expression levels of Col1a1, E-cadherin, DRP1, MFN2, PKM2, vimentin and α-SMA in MLE12 cells from each group. (B) Immunofluorescence analysis of PKM2, DRP1 and MFN2 in MLE12 cells from each group. Scale bar = 30 μm (original magnification 600×). The values are presented as the means ± standard deviations, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. the control group; ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. the LPS group. LPS: lipopolysaccharide; Col1a1: collagen type I; α-SMA: alpha smooth muscle actin; PKM2: pyruvate kinase M2; DRP1: dynamin-related protein-1, MFN2: mitofusin-2

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany) for detection with specific primary antibodies including antibodies against collagen I (Col1a1, ab270993, Abcam), E-cadherin (14472, CST), alpha smooth muscle actin (α-SMA, A1011, ABclonal), vimentin (A19607, ABclonal), PKM2 (60268- 1-Ig, Proteintech), DRP1 (NB110-55288, Novus), and MFN2(9482 S, CST), followed by incubation with goat anti-rabbit (A0208, Beyotime) and anti-mouse (A0216, Beyotime) secondary antibodies.

Techniques: Inhibition, Concentration Assay, In Vitro, Expressing, Immunofluorescence, Control

Journal: Journal of translational medicine

Article Title: Pyruvate kinase M2 modulates mitochondrial dynamics and EMT in alveolar epithelial cells during sepsis-associated pulmonary fibrosis.

doi: 10.1186/s12967-025-06199-7

Figure Lengend Snippet: Fig. 5 Inhibition of PKM2 alleviates sepsis-associated pulmonary fibrosis. Shikonin was used in the animal model. (A) Analysis of mitochondrial morphol ogy in the lung tissue of each group by transmission electron microscopy. Scale bars = 20 μm (original magnification 3000×) and 5 μm (original magnifi cation 8000×). (B) Protein expression levels of PKM2, DRP1 and MFN2 in the lung tissue of each group. (C) Immunofluorescence analysis of PKM2, DRP1, MFN2 and E-cadherin in the lung tissue of each group. Scale bar = 40 μm (original magnification 500×). The values are presented as the means ± standard deviations, ***P < 0.001, ****P < 0.0001, vs. control group, ####P < 0.0001 vs. LPS group. LPS: lipopolysaccharide; PKM2: pyruvate kinase M2; DRP1: dynamin- related protein-1; MFN2: mitofusin-2

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany) for detection with specific primary antibodies including antibodies against collagen I (Col1a1, ab270993, Abcam), E-cadherin (14472, CST), alpha smooth muscle actin (α-SMA, A1011, ABclonal), vimentin (A19607, ABclonal), PKM2 (60268- 1-Ig, Proteintech), DRP1 (NB110-55288, Novus), and MFN2(9482 S, CST), followed by incubation with goat anti-rabbit (A0208, Beyotime) and anti-mouse (A0216, Beyotime) secondary antibodies.

Techniques: Inhibition, Animal Model, Transmission Assay, Electron Microscopy, Expressing, Immunofluorescence, Control

Journal: Journal of translational medicine

Article Title: Pyruvate kinase M2 modulates mitochondrial dynamics and EMT in alveolar epithelial cells during sepsis-associated pulmonary fibrosis.

doi: 10.1186/s12967-025-06199-7

Figure Lengend Snippet: Fig. 7 Pyruvate kinase M2(PKM2) modulates mitochondrial dynamics and epithelial-mesenchymal transition (EMT) in alveolar epithelial cells during sepsis-associated pulmonary fibrosis. LPS-induced upregulation of PKM2 increases DRP1 and decreases MFN2, disrupting mitochondrial dynamics, and leading to increased mitochondrial fragmentation and accelerated EMT, ultimately contributing to sepsis-associated pulmonary fibrosis

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany) for detection with specific primary antibodies including antibodies against collagen I (Col1a1, ab270993, Abcam), E-cadherin (14472, CST), alpha smooth muscle actin (α-SMA, A1011, ABclonal), vimentin (A19607, ABclonal), PKM2 (60268- 1-Ig, Proteintech), DRP1 (NB110-55288, Novus), and MFN2(9482 S, CST), followed by incubation with goat anti-rabbit (A0208, Beyotime) and anti-mouse (A0216, Beyotime) secondary antibodies.

Techniques:

Journal: Antioxidants

Article Title: Pimozide and Imipramine Blue Exploit Mitochondrial Vulnerabilities and Reactive Oxygen Species to Cooperatively Target High Risk Acute Myeloid Leukemia

doi: 10.3390/antiox10060956

Figure Lengend Snippet: IB suppresses phosphorylation of DRP1 in Flt3-ITD + cells. ( A ) A panel of genes related to the ER–mitochondria interface were assayed by real-time qRT-PCR. The panel included Fis1, Ip3r, Mfn1, Drp1, Grp75, Mtorc2, Vdac1, and Bap31 ( n = 3 for all except Drp1 where n = 7). p values were calculated relative to OCI-AML3 cells. ( B ) Western blot analysis for total Drp1 protein with β-actin as the loading control. ( C ) Western blot analysis for phospho-Drp1 protein with β-actin as the loading control. ( D , E ) Densitometry analysis was performed and quantitation is shown for replicates of the Western blots in panels C and D. n = 3 and p values were calculated with comparisons as shown between either OCI-AML3 cells or with or without treatment. * p < 0.05; ** p < 0.01.

Article Snippet: Antibodies for Western blot were obtained from the following: Anti-STAT5 (Phospho-STAT5 Y694) antibody (Abcam, Cambridge, UK), Phospho-Akt (Ser473) (D9E), Phospho-Akt (Thr308) (D25E6) (Cell Signaling), Phospho-DRP1 (Ser637) (D3A4) (Cell Signaling, Danvers, MA, USA), DRP1 Antibody (Novus Biologicals), p38alpha MAPK antibody and Phospho-p38alpha MAPK (Cell Signaling, Danvers, MA, USA).

Techniques: Phospho-proteomics, Quantitative RT-PCR, Western Blot, Control, Quantitation Assay