Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on total levels of proteins that regulate mitochondrial dynamics. (A) Mfn1 and (B) DRP1 western blot of lysates from PC12 cells treated with FCCP (10 μM, 2 h) and AβOs (0.5 μM, 1 and 2 h). A dotted line on the western blot indicates different regions of the same gel. (C, D) Quantification of Mfn1 and DRP1 levels normalized to beta actin. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 compared between the control group. One-way ANOVA with the Dunnett's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n = 4–5 for each group) (original gel blot are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on immunoreactivity of proteins that regulate mitochondrial dynamics. Representative epifluorescence images of (A) Mfn1 and (B) DRP1 immunoreactivity in PC-12 cells control and treated with AβOs (0.5 μM) for 1 and 2 h. Quantification of (C) Mfn1 and (D) DRP1 immunoreactivity (intensity), under the same experimental conditions. Scale bars: 20 μm. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001 compared between the control group. One-way ANOVA with the Dunn's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n=3–6 for each group, N= 56–103) (entire inmunocytochemistry for Mfn1 and DRP1 with control are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Model of alterations induced on SIRT1/PGC-1α pathway by AβOs. (A) . Healthy neuronal conditions were energy depletion and/or decreased catabolic rates can be sensed by SIRT1 promoting the PGC-1α deacetylation (1). Transcription and co-activates of factors like NRF- 1/2 for the expression of nuclear-encoded mitochondrial genes and dynamic mitochondrial proteins, requires of PGC-1α translocation (2). Coordinated mitochondrial dynamics (3, fission/fusion), depends of adequate expression of Mfn1, Drp1. (B) . In AβOs treated neurons, the PGC-1α is unable to be deacetylated and to translocate to the nucleus (4). The expression of key genes is loss (5), and imbalance between fusion and fission to promote the granular mitochondrial phenotype (6) and neuronal death.

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Expressing, Translocation Assay

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

Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: Summary of quantitative real-time RT-PCR oligonucleotide primers used in measuring mRNA expression in mitochondrial dynamics, mitochondrial biogenesis, and synaptic genes in in N2a cells treated with Aβ42, Mdivi1+Aβ42, Aβ42+Mdivi1 relative to untreated N2a cells

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques: Quantitative RT-PCR, Expressing, Sequencing

Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, and synaptic proteins in N2a cells treated with Aβ42, Mdivi1+Aβ42, Aβ42+Mdivi1 relative to untreated N2a cells

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques: Western Blot

Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: mRNA fold changes in N2a cells treated with Aβ42 and Mdivi1

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques:

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: A cGAS and DRP1 expression levels after 6 hours of culture of PMA-differentiated human THP1 macrophages depleted or not for DRP1, and irradiated (or not) with a single dose of 2 Gy. Representative westerns are shown. GAPDH is used as loading control. B, C Confocal microscopy images ( B ) and percentages ( C ) of PMA-differentiated human THP1 macrophages showing iNOS ( B,C ) and ψ-H2AX ( C ) expressions 24 hours after treatment with control, 2 Gy single-dose IR, 5 μM G140, or 2 Gy single-dose IR combined with 5 μM G140 are shown. Nuclei are stained with Hoechst 33342 (scale bar, 5 μm). Images are representative from 3 independent experiments. D IFR5 expression levels detected after 96 hours of culture of PMA-differentiated human THP1 macrophages that have been treated with control, 2 Gy single-dose IR, 5 μM G140, or 2 Gy single-dose IR combined with 5 μM G140. Representative western blots are shown. GAPDH is used as loading control. E IFR5 expression levels detected after 6 hours of culture of murine RAW264.7 macrophages that have been treated with control, 2 Gy single-dose IR, 10 μM RU521, or 2 Gy single-dose IR combined with 10 μM RU521. Representative western blots are shown. GAPDH is used as loading control. F cGAS expression after 96 hours of transfection with control and cGAS-specific siRNA (sicGAS). Representative western blots are shown. GAPDH is used as loading control. G IRF5 expression levels detected after 96 hours of culture of PMA-differentiated human THP1 macrophages depleted or not for cGAS and treated with control or 2 Gy single-dose irradiation. Representative western blots are shown. GAPDH is used as loading control. Data are means ± S.E.M from three independent experiments. P-values (**P< 0.01 and ***P< 0.001) were determined with two-way ANOVA with Tukey’s multiple comparisons test ( C ).

Article Snippet: For western blots, anti-cGAS (D1D3G) (#15103), anti-cGAS (D3O8O) (#31659), anti-Phospho-DRP1 (Ser616) (#3455) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Irradiation, Control, Confocal Microscopy, Staining, Western Blot, Transfection