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: 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