Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: Model structures of HexA (PDB:2GJX) and HexB (PDB:1NOU) sub-unit proteins, highlighting the location of pathogenic mutations. Also shown autophagy in fibroblasts obtained in culture from patients with GM2 gangliosidosis (Tay–Sachs and Sandhoff diseases). ( A ). HexA point mutations: different colours depict amino acid substitutions identified in the cognate structures identified in different mutations studied. ( B ). Frameshift mutations in the alpha subunits found in two patients with Tay–Sachs disease are shown in yellow and orange; premature stop codons are marked by an asterisk. ( C ). The surface of hexosaminidase A with the critical active site region required for hydrolysis of GM2 ganglioside (CRH_GM2). The propeptide is shown in grey and the mature protein chain is depicted in white. ( D ). Enzymatic activity of HexA in fibroblast homogenates. ( E ). Morphological changes in fibroblasts from Tay–Sachs patients compared with control cells. ( F ). Cell growth determined in healthy and Tay–Sachs fibroblasts. ( G ). Expression of autophagy proteins in control and Tay–Sachs fibroblasts: LC3-I (top panels, top band), LC3-II (top panels, bottom band). ( H ). Immunofluorescence staining with anti-p62 antibody. ( I ). Impaired autophagic flux in Tay–Sachs fibroblasts. Determination of LC3-II in the presence and absence of bafilomycin A1 in control (CTL) and fibroblasts from Tay–Sachs patients; bafilomycin A1 was used at a final concentration of 100 nM with 12 h exposure. Total cellular extracts were analysed by immunoblotting with antibodies against LC3. The data are the mean ± SD for experiments conducted on two different control cell lines. Data represent the mean ± SD of three separate experiments. *** p < 0.001, ** p < 0.005, * p < 0.05 between cells from control subjects and patients with Tay–Sachs disease. a p < 0.05; aa p < 0.01; aaa p < 0.001.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Activity Assay, Control, Expressing, Immunofluorescence, Staining, Concentration Assay, Western Blot

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: Expression of mTOR and AKT protein were determined in cultured control and Tay–Sachs disease fibroblasts. Data represent the mean ± SD of three separate experiments.* p < 0.05; ** p < 0.01; *** p < 0.001 between transfected and non-transfected cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Cell Culture, Control, Transfection

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A ). Expression of LC3, p62, CatB, CatD, mTOR and AKT proteins determined in human control and Sandhoff disease fibroblasts. ( B , C ). Immunofluorescence of CatB in control and pathological cells with quantification in Sandhoff disease fibroblasts. ( B , D ). Characteristic ultrastructure with altered autophagosome abundance quantified in Sandhoff disease fibroblasts. ( E ). Expression of LC3, p62, CatB and mTOR proteins in the brain and spinal cord obtained from wild type and hexb −/− mutant mice with GM2 gangliosidosis (Sandhoff disease). Densitometry results are presented as means ± SEM, n = 10 mice. * p < 0.05; ** p < 0.01; *** p < 0.001 between control and diseased fibroblasts and wild type and hexB −/− mutant mice.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Control, Immunofluorescence, Mutagenesis

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A ). Expression of mTOR and AKT determined in control and representative Tay–Sachs fibroblasts after L-arginine treatment. ( B ). Protein synthesis was quantified in extracts of control and Tay–Sachs fibroblasts treated with L-arginine using puromycin labeling followed by immunoblotting. ( C ). Representative image of Tay–Sachs treated fibroblasts after transfection of the mCherry-GFP-LC3 plasmid and quantification of autophagic puncta. For control cells, the data are the mean ± SD for experiments conducted on two different control cell lines. GAPDH was used as a loading control. Data represent the mean ± SD of three separate experiments. *** p < 0.001 between control and Tay–Sachs fibroblasts; aa p < 0.01; aaa p < 0.001 between non-treated and treated cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Control, Labeling, Western Blot, Transfection, Plasmid Preparation

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A , B ). Immunofluorescence of CatB and HexA in control and Sandhoff disease fibroblasts and quantification after L-arginine treatment. ( C ). Expression of CatB and HexA protein were determined in control and representative Tay–Sachs fibroblast cultures after L-arginine treatment in vivo. ( D ). Expression of mTOR, CatB, and ASS1 (arginosuccinate synthetase) proteins was determined in peripheral blood mononuclear cells obtained from a patient with juvenile Tay–Sachs disease and a patient with juvenile Sandoff disease after oral L-arginine treatment. Data represent the mean ± SD of three separate experiments.* p < 0.05; ** p < 0.01; *** p < 0.001 between control and Tay–Sachs patients; a p < 0.05; aa p < 0.01; aaa p < 0.001 between non-treated and treated cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Immunofluorescence, Control, Expressing, In Vivo

Journal: Cells

Article Title: Impact of Progerin Expression on Adipogenesis in Hutchinson—Gilford Progeria Skin-Derived Precursor Cells

doi: 10.3390/cells10071598

Figure Lengend Snippet: Isolation of SKPs from control and HGPS fibroblasts. ( a ) Panel showing the protocol for SKP isolation. Briefly, fibroblasts were pelleted and treated with HBSS buffer (pH 5.7) for 30 min at 37 °C. Cells were cultured in SKP media containing DMEM low glucose, EGF, FGF, and B27. The flasks were agitated daily, and the spheroids were harvested at day 4 for analysis. ( b ) SKP formation from both control (GMO1651c, GMO5565) and HGPS (HGADFN127, HGADFN003) fibroblasts with 5 and 30% senescence (SNS). ( c , d ) Quantification of the number and the diameter of the spheroids from control and HGPS fibroblast cultures with 5 and 30% SNS at day 4. Values are presented as mean ± SD ( n = 3), not significant (ns), * p < 0.05, ( c , d ) unpaired t -test. HBSS: Hank’s Balanced Salt Solution, DMEM: Dulbecco′s modified Eagle medium, EGF: epidermal growth factor, FGF: fibroblast growth factor, SKPs: skin-derived precursor cells, SNS: senescence.

Article Snippet: The human primary dermal fibroblast cell lines, GMO5565 (3-year-old male), GMO1651 (13-year-old female) and GMO5567A (12-year-old male) were all obtained from the Coriell Institute for Medical Research (Camden, NJ, USA).

Techniques: Isolation, Control, Cell Culture, Modification, Derivative Assay