Journal: Journal of medicinal chemistry

Article Title: Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome

doi: 10.1021/acs.jmedchem.1c00419

Figure Lengend Snippet: Polyamine levels in wild-type (a: CMS-24949) and SMS mutant (b: CMS-26559, c: CMS-6233, and d: CMS-23916) fibroblast cell lines with or without treatment of Spm or prodrug 1 (5 μM) in the presence of AG (1 mM) after 72 h incubation at 37 °C. Values represent data from the experiment performed in triplicates ± SD, *p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The authors wish to thank Dr. Charles Schwartz at the Greenwood Genetic Center for supplying the SRS cell lines and control wt fibroblasts and the Snyder Robinson Foundation for financial support of this work (UCF25068A04 to O.P.).

Techniques: Mutagenesis, Incubation

Journal: Journal of medicinal chemistry

Article Title: Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome

doi: 10.1021/acs.jmedchem.1c00419

Figure Lengend Snippet: Polyamine levels in CMS-23916 (mutant) fibroblast cell lines untreated (UT) or treated with prodrug 1 (5 μM) only, NAC (2 mM) only, or a combination of prodrug 1 (5 μM) and NAC (2 mM). Values represent data from the experiment performed in triplicates ± S.D. *p < 0.05.

Article Snippet: The authors wish to thank Dr. Charles Schwartz at the Greenwood Genetic Center for supplying the SRS cell lines and control wt fibroblasts and the Snyder Robinson Foundation for financial support of this work (UCF25068A04 to O.P.).

Techniques: Mutagenesis

Journal: Orphanet Journal of Rare Diseases

Article Title: Arabidopsis thaliana alternative dehydrogenases: a potential therapy for mitochondrial complex I deficiency? Perspectives and pitfalls

doi: 10.1186/s13023-019-1185-3

Figure Lengend Snippet: Oxygen consumption analysis: Oxygen consumption was evaluated using the Seahorse XF Analyzer; a Oxygen consumption rate (OCR) expressed as percent (%) of rate measurement 13 in control cells (NDHF) and in control cells transduced with alternative dehydrogenases from A. thaliana (+AtNDA1, +AtNDA2, +AtNDB4) and yeast (+MTS-ScNDI1); b OCR expressed as % of rate measurement 13 in NDHF, in NDUFS4-deficient cells (NDUFS4) and in patient cells transduced with alternative dehydrogenases from A. thaliana (+AtNDA1,+AtNDA2, +AtNDB4) and yeast (+MTS-ScNDI1); c Maximal respiration rate in CI deficient cells (carrying pathogenic variants in ACAD9 and NDUFS4), before and after transduction with AtNDA1, AtNDA2, AtNDB4 and MTS-ScNDI1. Values were normalized to maximal respiration of untransduced control cells; d Oxygen consumption rate (OCR) expressed as pmol O 2 /min/cell in cell lines presenting with CI-defect due to mutations in ACAD9, NDUFB3, NDUFS4, ND5, tRNA Leu before and after transduction with alternative dehydrogenases from A. thaliana and yeast. Each cell line was measured at least twice in independent experiments. During experiment, four technical replicates were run for each cell line. Values are expressed as mean ± SD

Article Snippet: The control fibroblasts (NDHF) were purchased from Lonza (Cat. No. CC-2509).

Techniques: Control, Transduction

Journal: Orphanet Journal of Rare Diseases

Article Title: Arabidopsis thaliana alternative dehydrogenases: a potential therapy for mitochondrial complex I deficiency? Perspectives and pitfalls

doi: 10.1186/s13023-019-1185-3

Figure Lengend Snippet: Assessment of SOD activity: Spectrophotometric assessment of SOD activity according to the pyrogallol autoxidation method. NDUFS4 mutated cell line (79787) displays higher SOD activity when compared to control fibroblasts (NHDF). Transfection with AtNDB4 and AtNDA2 (79787-AtNDB4 and 79,787-AtNDA2) significantly decreases SOD activity, which is nearly restored to normal levels. Values are expressed as means ± SD (ns: not significant; *p < 0.05; **p < 0.01 )

Article Snippet: The control fibroblasts (NDHF) were purchased from Lonza (Cat. No. CC-2509).

Techniques: Activity Assay, Control, Transfection

Journal: Orphanet Journal of Rare Diseases

Article Title: Arabidopsis thaliana alternative dehydrogenases: a potential therapy for mitochondrial complex I deficiency? Perspectives and pitfalls

doi: 10.1186/s13023-019-1185-3

Figure Lengend Snippet: Evaluation of mitochondrial respiration: Mitochondrial membrane potential variations assessed by rhodamine 123 fluorescence and oxygen uptake measured with optode device in digitonine permeabilized fibroblasts (representative graphs for the control fibroblasts - left panel and AtNDA2-transfected control fibroblasts - right panel). The reaction was started by the addition of glutamate/malate, followed by injections of ADP (see text). Note that the amount of oxygen reduced during ADP phosphorylation is significantly higher in AtNDA2 transfected cells comparing to control

Article Snippet: The control fibroblasts (NDHF) were purchased from Lonza (Cat. No. CC-2509).

Techniques: Membrane, Fluorescence, Control, Transfection, Phospho-proteomics

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of RPE-1 nuclei mock treated and treated with myriocin for 24 h. Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. (B) Percentage of nuclear abnormalities of RPE-1 cells treated with myriocin (n = 100 cells). (C) Representative images of RPE-1 nuclei upon knockdown of SPTLC1 or SPTLC2 after 72 h. (D) Percentage of nuclear abnormalities of RPE-1 cells upon knockdown of SPTLC1 or SPTLC2 (n = 100 cells). (E) Western blot analysis of SPTLC1 and SPTLC2 protein levels in RPE-1 cells. (F) Representative images of RPE-1 nuclei treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (Cer) for 24 h. (G) Percentage of RPE-1 cells showing abnormal nuclear morphology when treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (n = 100 cells). (H) Representative images of primary human skin fibroblast (HSF) nuclei mock treated and treated with myriocin for 24 h. (I) Percentage of nuclear abnormalities of primary HSF treated with myriocin (n = 100 cells). (J) Representative images of primary HSF nuclei treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide for 24 h. (K) Percentage of primary HSF showing abnormal nuclear morphology when treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (Cer) (n = 100 cells). All images are representative of three biological replicates. Scale bars (A, C, F, H, and J), 10 μm.

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Staining, Knockdown, Western Blot

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of primary human fibroblasts from 2 euploid donors and 2 patients with Down syndrome. Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. Scale bar, 2.5 μm. (B) Percentage of fibroblasts from euploid donors showing abnormal nuclear morphology when treated with fumonisin B 1 or SKi-II (n = 100). (C) Percentage of fibroblasts from 2 patients with Down syndrome showing abnormal nuclear morphology treated with fumonisin B 1 , SKi-II, or ceramide (Cer) (n = 100). (D) Representative images of trisomy 21 nuclei treated with fumonisin B 1 , SKi-II, or ceramide. Scale bar, 10 μm. (E) Representative images of primary human fibroblasts from 2 euploid donors and 2 patients with Patau syndrome(trisomy 13) or Edward syndrome (trisomy 18). Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. Scale bar, 2.5 μm. (F) Percentage of fibroblasts from 2 patients with Patau syndrome showing abnormal nuclear morphology treated with fumonisin B1, SKi-II, orceramide (n = 100). (G) Percentage of fibroblasts from 2 patients with Edward syndrome showing abnormal nuclear morphology treated with fumonisin B1, Ski-II, or ceramide (n = 100).

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Staining

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of primary human fibroblasts from euploid donors and patients with Patau syndrome (trisomy 13), Edward syndrome (trisomy 18), or Down syndrome (trisomy 21). Immunofluorescence is shown for lamin A/C, H3K9triMe, and Hoechst 33342. In the merge images, lamin A/C is green, H3K9triMe is red, and DNA is blue. Scale bar, 5 μm. (B) Western blot analysis of H3K9triMe in human fibroblasts. (C) Quantification of the number of 53BP1 foci in human fibroblasts mock treated or in the presence of fumonisin B 1 (n = 100). (D) Representative images of primary human fibroblasts from euploid donors and patients with Down syndrome. Immunofluorescence is shown for 53BP1 (red) and Hoechst 33342 (blue). There is no correlation between abnormal nuclear morphology and number of 53BP1 foci. Scale bar, 5 μm. (E) Growth curves of primary human fibroblasts from 2 euploid donors and 2 patients with Patau (trisomy13), Edward (trisomy18), or Down syndrome(trisomy 21) with increasing concentrations of fumonisin B 1 . (F) Representative images of primary human astrocytes from 4 euploid donors and 4 donors with Down syndrome. Immunofluorescence for the nucleus is blue stained with Hoechst 33342. Scale bar, 5 μm. (G) Percentage of astrocytes from euploid and trisomy 21 donors showing abnormal nuclear morphology (n > 200).

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Western Blot, Staining

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet:

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Recombinant, Membrane, Protease Inhibitor, Control, Software