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: bioRxiv

Article Title: Interactions between the N- and C- termini of mechanosensitive ion channel At MSL10 are consistent with a three-step mechanism for activation

doi: 10.1101/726521

Figure Lengend Snippet: (A) Elevated levels of H 2 O 2 detected by DAB staining. Representative bright-field images of rosette leaves are shown. Bar = 200 μm. (B) Colorimetric quantification of NBT-formazan deposition on rosette leaves. FW, fresh weight. Data are means ± SD of three replicates (n=18). (C) Hydrogen peroxide (H 2 O 2 ) content, quantified as the H 2 O 2 (mmol) per gram dry weight (DW) of rosette leaves using Amplex Red-coupled fluorescence quantitative assay. Values are means ± SD of three replicates (n=14). (A), (B) , and (C) Leaves from three-week-old plants from each line grown at 21°C (top row) under 24-h light regime were used for the experiment. Three independent T3 homozygous transgenic lines expressing MSL10g, MSL10g 7A or MSL10g 7D in the msl10-1 background were selected for comparison.

Article Snippet: For the quantitative measurement of hydrogen peroxide concentrations, the Amplex Red Hydrogen/Peroxidase Assay Kit (Molecular Probes, Invitrogen) was used, following the manufacturer’s instructions and as described in ( ).

Techniques: Staining, Fluorescence, Transgenic Assay, Expressing