Journal: bioRxiv

Article Title: mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts

doi: 10.1101/2022.02.21.479058

Figure Lengend Snippet: (A) Overview of the rationale behind mRNA-based treatment of NP-C1 disease. (B) NPC1 and Hoechst 33342 staining in healthy fibroblasts (GM03652), Lipofectamine-treated patient fibroblasts (GM18393), and GM18393 patient fibroblasts treated with 50 ng of M1ψTP-modified NPC1 mRNA. Cells were seeded at a density of 3000 per well and imaged 24 h after treatment. Images are representative of three independent experiments. (C) Quantification of NPC1 staining from the experiment in (B), as measured by average NPC1 fluorescence per cell (cumulative NPC1 fluorescence was divided by the total number of cells identified via Hoescht staining, using the PerkinElmer Harmony high-content analysis software). Data are mean ± s.d. of three technical replicates (from one representative of three independent experiments), encompassing 7 imaging fields per well and >300 cells per treatment condition. (D) Quantification of total and unesterified cholesterol levels in healthy cells, Lipofectamine-treated GM18393 cells, and GM18393 cells treated with 50 ng NPC1 M1ψTP mRNA, after a 48 h incubation in lipoprotein-deficient serum followed by either a 6 h incubation with 50 μg/ml LDL or a further 6 h incubation with lipoprotein-deficient serum to establish a baseline control. Total and unesterified cholesterol levels were assayed using the Amplex Red Cholesterol Assay Kit. Data are mean ± s.d. of two independent experiments. (E) Filipin and Lysotracker Red DND-99 staining in healthy cells, Lipofectamine-treated GM18393 cells, and 50 ng mRNA-treated GM18393 cells after a 48 h incubation in lipoprotein-deficient serum followed by 6 h incubation with 50 μg/ml LDL. Images are representative of three independent experiments. (F) Quantification of filipin staining from the experiment in (E), as measured by average filipin fluorescence per well. Data are mean ± s.d. of three technical replicates (from one representative of three independent experiments), encompassing 9 imaging fields per well and >600 cells per treatment condition. (G) Quantification of lysosome size from Lysotracker Red staining in (E), using the PerkinElmer Harmony high-content analysis software. Data are mean ± s.d. of two technical replicates (from one representative of three independent experiments), encompassing 9 imaging fields per well and >300 cells per treatment condition. In (C), (F) and (G), * P < 0.05, ** P < 0.01 by ordinary one-way ANOVA followed by a Dunnett’s multiple comparisons test. In (D), **** P < 0.0001 by two-way ANOVA followed by a Tukey’s multiple comparisons test.

Article Snippet: After a further 6 h, intracellular levels of unesterified cholesterol and total cholesterol were separately assayed using the Amplex Red Cholesterol Assay Kit (Promega), following the manufacturer’s instructions.

Techniques: Staining, Modification, Fluorescence, High Content Screening, Software, Imaging, Incubation, Amplex Red Cholesterol Assay

Journal: PLoS ONE

Article Title: Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

doi: 10.1371/journal.pone.0070407

Figure Lengend Snippet: Biochemical characteristics of mammary gland derived enriched plasma membranes (EPM).

Article Snippet: The Amplex Red Cholesterol Assay kit, antibiotics, and antimycotics were purchased from LubioScience (Luzern, Switzerland).

Techniques: Derivative Assay, Clinical Proteomics, Binding Assay, Inhibition

Journal: PLoS ONE

Article Title: Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

doi: 10.1371/journal.pone.0070407

Figure Lengend Snippet: The figure illustrates representative kinetics of incorporation of 1nM (●) and 10nM (■) 3 H-cholesterol into EPM (100µg) isolated from lactating MG tissues. Data represent the means of three independent experiments performed in triplicates. The incorporation reaction was incubated at 37°C using glass tubes coated with bovine serum albumin. The radioactivity of the filter was measured using a β-counter. No difference was found between lactating and non-lactating MG.

Article Snippet: The Amplex Red Cholesterol Assay kit, antibiotics, and antimycotics were purchased from LubioScience (Luzern, Switzerland).

Techniques: Isolation, Incubation, Radioactivity

Journal: PLoS ONE

Article Title: Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

doi: 10.1371/journal.pone.0070407

Figure Lengend Snippet: A : Comparative uptake (●) and efflux (▲) of 3 H-cholesterol by MeBo cells growing as a monolayer in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% antibiotics/antimycotics. Cholesterol efflux was performed in the presence of 10µg/ml apoA-I (details see Materials and Methods). The cholesterol uptake was calculated either based on the amount of radiolabel disappearing from the medium (evaluation 1) or on the sum of the radiolabel measured in the cell lysate and efflux medium (evaluation 2). Both values were related to the initially loaded amounts of radiolabel that was defined as 100%. The arrow depicts the inversion point that is the incubation time where cholesterol uptake and efflux are in apparent equilibrium. It represents a threshold beyond which the availability of 3 H-cholesterol for efflux becomes markedly reduced in favor of increasing intracellular compartmentalization.” B : Cholesteryl ester content of the cell lysate. Cholesteryl esters were measured with the Amplex Red ® assay kit according to the manufacturer’s instructions. All experimental details were as described in section A. C : Time-dependent saturation curve of apoA-I mediated efflux. Cells were loaded with cholesterol for 0.5h (●), 1h (■), and 24h (▲). Details for cell equilibration were as described in section A. Please note that in contrast to , the background efflux measured in the absence of apoA-I was recorded, and subtracted from the total efflux measured in the presence of 10µg/ml of apoA-I. D : Regulation of apoA-I mediated efflux in MeBo cells. Cells were loaded with 3 H-cholesterol (1µCi/ml) in complete DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% antibiotics for 24h. Cells were equilibrated for 18h in serum-free medium followed by the efflux in the presence of apoA-I (10µg/ml) for 4h (see Materials and Methods for additional details). Cells were treated with probucol, an inhibitor of ABCA1, throughout the efflux time. All data are expressed as means ± SD of three independent experiments performed in triplicates.

Article Snippet: The Amplex Red Cholesterol Assay kit, antibiotics, and antimycotics were purchased from LubioScience (Luzern, Switzerland).

Techniques: Incubation, Amplex Red Assay

Journal: PLoS ONE

Article Title: Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

doi: 10.1371/journal.pone.0070407

Figure Lengend Snippet: Comparative 3 H-cholesterol uptake and efflux in primary bovine mammary epithelial cells.

Article Snippet: The Amplex Red Cholesterol Assay kit, antibiotics, and antimycotics were purchased from LubioScience (Luzern, Switzerland).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

doi: 10.1371/journal.pone.0070407

Figure Lengend Snippet: Resistance was measured according to the manufacturer’s instructions in quadruplicates of >12 wells. Trans-epithelial electrical resistance was calculated according to . B: Vectorial apoA-I mediated 3 H-cholesterol efflux in MeBo cells. The experiment was performed according to the optimized protocol (loading 1h, equilibration 1h, efflux 1h). All other details of the procedure were as described in . ApoA-I was added either to the apical (A) or to the basal (B), or to both chambers (A’, B’). ApoA-I mediated cholesterol efflux was calculated separately for the apical and the basal chamber by subtracting the background efflux. All data are expressed as means ± SD of triplicates measurements.

Article Snippet: The Amplex Red Cholesterol Assay kit, antibiotics, and antimycotics were purchased from LubioScience (Luzern, Switzerland).

Techniques: