Journal: EMBO Molecular Medicine

Article Title: Unraveling autophagic imbalances and therapeutic insights in Mecp2-deficient models

doi: 10.1038/s44321-024-00151-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: HCS Studio software using SpotDetector bioapplication , Thermo Fisher Scientific , .

Techniques: Control, Mutagenesis, Recombinant, Sequencing, Protease Inhibitor, Saline, Electron Microscopy, Western Blot, Software, Microscopy, Transmission Assay

Journal: Scientific Reports

Article Title: High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

doi: 10.1038/s41598-024-64366-0

Figure Lengend Snippet: Schematic of High Content Analysis (HCA) image processing and analysis optimisation. To analyse the fluorescence intensity of EGFP-/tGFP- and tdTomato (tdT)/mCherry-fusion proteins and quantify protein inclusions containing fluorescent fusion proteins in NSC-34 cells, we deployed the Spot Detector BioApplication designed to analyse fluorescent foci in cells. Optimisation was carried out using images of NSC-34 cells triple-transfected to express H2B-ECFP, either SOD1 WT -EGFP, SOD1 A4V -EGFP, TDP-43 WT -tGFP TDP-43 M337V -tGFP, FUS WT -tGFP, FUS R495X -tGFP, FUS R521G -tGFP or EGFP alone and mCherry alone. Cells were imaged at 48 h post-transfection using a 20 × objective lens. ( a ) Raw images from Channels 1 (H2B-ECFP), 2 (EGFP-/tGFP-fusion proteins) and 3 (tdT/mCherry-fusion proteins) were first pre-processed to remove background fluorescence, exclude cells positioned on the border of each image from analysis and distinguish individual cells (‘object’ segmentation). Channel 1 images were additionally smoothed (blurred) to help reduce fluorescent noise that could lead to the false inclusion of image artefacts in subsequent analyses. ( b ) Biological ‘objects’, in this case cells, were identified using nuclear-localised H2B-ECFP fluorescence in Channel 1 images. To select viable transfected cells for analysis and exclude image artefacts, dead cells and cell debris, cells were selected based on the size and fluorescence intensity of their ECFP-fluorescent nuclei. ( c ) The relevant measures for GFP fluorescence intensity and fluorescent foci were measured in Channel 2 within a circular analysis mask that expanded the mask derived in Channel 1. The green circular mask indicates cells selected for analysis, while yellow masks indicate fluorescent foci/‘spots’ selected for analysis. To detect and analyse fluorescent foci corresponding to protein inclusions, upper and lower limits for size and fluorescence intensity were set. ( d ) Channel 3 objects were identified using the same mask as Channel 2.

Article Snippet: Phase contrast and fluorescent images from 20 fields of view per well were acquired, with image analysis parameters optimised using the SpotDetector V4 BioApplication in HCS Studio (Thermo Scientific) summarised in Fig. (further detail in Figure ).

Techniques: High Content Screening, Fluorescence, Transfection, Derivative Assay

Journal: Scientific Reports

Article Title: High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

doi: 10.1038/s41598-024-64366-0

Figure Lengend Snippet: Optimisation of High Content Analysis of Fluc-EGFP foci in transfected cells. Representative Cellomics ® ArrayScan™ VTI images showing SpotDetector BioApplication masks (first and third rows of each panel) used to identify and select NSC-34 cells co-transfected with either ( a ) SOD1 WT -tdTomato, ( b ) SOD1 A4V -tdTomato, ( c ) CCNF WT -mCherry or ( d ) CCNF S621G -mCherry and Fluc WT -EGFP, Fluc SM -EGFP or Fluc DM -EGFP. Cells were imaged at 48 h post-transfection. Green circular masks indicate cells selected for analysis, yellow masks indicate ‘spots’ selected for analysis, representing aggregates. Images were acquired using a 20 × objective lens.

Article Snippet: Phase contrast and fluorescent images from 20 fields of view per well were acquired, with image analysis parameters optimised using the SpotDetector V4 BioApplication in HCS Studio (Thermo Scientific) summarised in Fig. (further detail in Figure ).

Techniques: High Content Screening, Transfection

Journal: Scientific Reports

Article Title: High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

doi: 10.1038/s41598-024-64366-0

Figure Lengend Snippet: HCA analysis of Firefly luciferase mutants reports on chaperone network activity in NSC-34 cells expressing SOD1 and CCNF. ( a ) Numbers of Fluc-EGFP aggregates per 100 transfected cells, ( b ) mean size of Fluc-EGFP aggregates (µm 2 ) and ( c ) mean fluorescence intensity (FI) of Fluc-EGFP aggregates imaged at 48 h post-transfection in NSC-34 cells expressing Fluc WT -EGFP, Fluc SM -EGFP or Fluc DM -EGFP with (i) mCherry alone ± treatment with 5 µM MG132, (ii) SOD1 WT -tdTomato or SOD1 A4V -tdTomato or (iii) CCNF WT -mCherry or CCNF S621G -mCherry. Treatment with MG132 was carried out at 30 h post-transfection. For mock treatment, 5 µM DMSO was instead added to cells. Graphs represent the mean ± SEM from quadruplicate wells of cells in n = 1 experiment, analysed using Cellomics ® ArrayScan™ VTI and SpotDetector BioApplication. Differences between the means were determined using One-Way ANOVA followed by Tukey’s Multiple Comparison Test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. ( d ) Representative confocal images of Hoechst-stained NSC-34 cells expressing Fluc WT -EGFP, Fluc SM -EGFP or Fluc DM -EGFP with (i) SOD1 WT -tdTomato or SOD1 A4V -tdTomato or (ii) CCNF WT -mCherry or CCNF S621G -mCherry. Scale bars represent 10 µm.

Article Snippet: Phase contrast and fluorescent images from 20 fields of view per well were acquired, with image analysis parameters optimised using the SpotDetector V4 BioApplication in HCS Studio (Thermo Scientific) summarised in Fig. (further detail in Figure ).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Fluorescence, Comparison, Staining