HEK293 arrestin-2/3 KO cells were co-transfected with HA-ASK1, HA-JNK3α2 and either control (Venus) or indicated N-terminally Venus-tagged forms of arrestin-3 (to mimic the conditions of the JNK activation). Twenty-four-hour post-transfection the cells were replated onto poly-D-lysine- and fibronectin-coated Mattek glass bottom dishes. The next day, the medium in the dishes was replaced with 2 ml/dish of FluoroBrite, and the cells’ nuclei were stained with NucBlue reagent (ThermoFisher) according to the manufacturer’s instructions. The cells were then imaged live on the Olympus confocal microscope. Images of between 25 and 45 cells were collected for the analysis with Venus and each form of arrestin-3. The images were analyzed with Nikon NIS-Elements software.
Nucblue reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
NucBlue reagent is a fluorescent DNA stain used for nuclear staining in cells. It binds to DNA and emits fluorescence when excited, allowing for the visualization of cell nuclei.
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Lab products found in correlation
Confocal microscope, by Olympus (2 mentions)
Nis elements software, by Nikon (2 mentions)
Cellmask deep red plasma membrane dye, by Thermo Fisher Scientific (2 mentions)
Sensoplate, by Greiner (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
X tremegene transfection reagent, by Roche (1 mentions)
Axiovision version 4, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Anti myc mab, by Santa Cruz Biotechnology (1 mentions)
Cellhesion 200, by Bruker (1 mentions)
Tubulin tracker green, by Thermo Fisher Scientific (1 mentions)
Phenol red free dmem f12, by Thermo Fisher Scientific (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Sp8x confocal microscope, by Leica (1 mentions)
Ds qi2 camera, by Nikon (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Eclipse ti2 fluorescent microscope, by Nikon (1 mentions)
Pluripotent stem cell 4 marker immunocytochemistry kit, by Thermo Fisher Scientific (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
25 protocols using nucblue reagent
1
Imaging of JNK Activation in HEK293 Cells
2
3D Cell Migration Tracking Protocol
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Imaging was made using an oil immersion objective with a magnification of 40× and a numerical aperture (NA) of 1.4. Cell Tracker v 1.0 Software with automatic vignetting and alignment, along with a semi-automated algorithm, was used to precisely track all cells in the field of view. For the identification of individual cells, a dynamic interpolation with histogram matching was set up. The tracking software algorithms were further optimized by using intravital staining of the cell nucleus with the NucBlue reagent from Thermo Fisher Scientific. This reagent, based on Hoechst 33342, is a commonly used cell-permeant nuclear counterstain that emits blue fluorescence upon binding to DNA. The measurement of the location of individual cells in 3 dimensions (X, Y, Z) was used to precisely determine all relevant migration parameters. The method used is an extension of the one described by Witko et al.8 (link), but in this case the “Z” direction was additionally taken into account in the analysis, illustrating the penetration of cells inside the materials internal 3D structure. The 3D migration analysis protocol is illustrated in Fig. 1 .
3
Imaging of JAK1 Mutant Cells
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U4C cells complemented with sfGFP-tagged JAK1 mutants were seeded at 50,000 cell per well on 24-well Sensoplates (662892, Greiner). Two days later, living cells were rinsed twice in 1xPBS and imaged in Live Cell Imaging Solution (A14291DJ, ThermoFisher). Nuclei were stained with NucBlue reagent (R37605, ThermoFisher). Micrographs were acquired at a Leica SP5 confocal microscope.
4
Visualizing Tsg101 Localization in HeLa Cells
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HeLa cells (ATCC CCL-2) grown on poly-l-lysine treated cover slips were transfected with pCMV-Gag-EGFP alone or together with pLLEXP1-hTsg101-myc using Roche X-tremeGene transfection reagent. After 48 h, cells were fixed in 4% formaldehyde (Thermo-Fisher, Waltham, MA, USA) and permeabilized in 0.1% Triton X-100/PBS. Tsg101 was detected in samples by indirect immunofluorescence using anti-myc Mab (Santa Cruz, CA, USA, SC-40, 1:100) and Texas Red tagged goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA, 1:400). Nuclei were stained with NucBlue reagent (Thermo Fisher). Z-stack images were captured on an inverted fluorescence/differential-interference contrast (dic) Zeiss Axiovert 200M deconvolving fluorescence microscope operated by AxioVision Version 4.5 software (Zeiss, Jena, Germany). Deconvolution image processing used the constrained iterative method (Zeiss AxioVision software). Protein co-localization was assessed in cells by determination of Pearson’s coefficient of correlation of the entire cell using Image J software (National Institutes of Health).
5
Measuring Nuclear Height Using AFM
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Focussed ion beam-sculpted cantilevers were fixed on a standard JPK glass block and mounted in the AFM head (CellHesion 200; JPK Instruments). Nuclei were stained with NucBlue® reagent (ThermoFischer Scientific). The bottom surface of the nucleus (attached to the glass) was focused and the cantilever approached to touch the glass surface close to the nucleus; this was set as 0 μm. The cantilever was retracted 20–25 μm away from the surface, positioned above the nucleus, and approached to touch the nucleus. Both, the piezo height and the force experienced by the cantilever, were monitored simultaneously in two different channels (Supplementary Fig. 20 ). Using the two signals and the point of cantilever deflection, the height of the nucleus was determined (Supplementary Fig. 20 ). Cantilever calibration was carried out using the thermal noise method (in-built calibration module).
6
Visualizing SRRM2-mCherry Induction
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The SRRM2-mCherry-inducible cell line was plated in four-well LabTek chamber slides. Day 1: cells were treated with 2 μg/ml Doxycycline to induce SRRM2-mCherry expression. Day 2: cells were treated with siCon or siNups as above. Day 4: cells were treated with one drop NucBlue reagent (ThermoFisher) per 2 ml of imaging media and 1 μM Tubulin Tracker Green (Thermofisher) 45 min prior to imaging in HEPES buffered, Phenol Red-free DMEM/F-12 (ThermoFisher) containing 10% FBS. Cells were imaged on a Leica SP8 White Light Nikon Ti-E widefield inverted microscope at 60× objective.
7
Arrestin-3 Modulation of JNK Activation
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HEK293 arrestin-2/3 KO cells were co-transfected with HA-ASK1, HA-JNK3α2, and either control (Venus) or indicated N-terminally Venus-tagged forms of arrestin-3 (to mimic the conditions of JNK activation). Twenty-four hours post-transfection, the cells were replated onto poly-D-lysine- and fibronectin-coated Mattek glass bottom dishes. The next day, the medium in the dishes was replaced with 2 mL/dish of FluoroBrite, and the cells’ nuclei were stained with NucBlue reagent (ThermoFisher) according to the manufacturer’s instructions. The cells were then imaged live on the Olympus confocal microscope. Images of between 25 and 45 cells were collected for the analysis with Venus and each form of arrestin-3. The images were analyzed with Nikon NIS-Elements software.
8
Cellular Metabolic Activity Profiling
9
Characterization of hiPSC and EB Cultures
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hiPSCs and EBs were seeded onto Geltrex-coated plastic coverslips (20012; SPL Life Sciences, Venice, Italy). Cells were washed twice with PBS, fixed with 4% PFA (158127; Sigma-Aldrich, Milan, Italy) for 30 min, permeabilized with 0.5% Triton X-100 (T8787; Sigma-Aldrich, Milan, Italy) in PBS for 15 min and blocked with 2% Normal Donkey Serum (ab7475; Abcam, Prodotti Gianni, Milan, Italy) + 0.2% Triton X-100 in PBS for 1h, all at RT. Cultures were then incubated with specific primary antibodies (Table 1 ) in blocking buffer overnight at 4 °C and stained for 1h with secondary antibody (Table 1 ) in blocking buffer at RT. Nuclei were counterstained using the NucBlue reagent (R37606; Thermo Fisher Scientific; Monza, Italy) for 10 min at RT. To assess the expression of pluripotency genes in hiPSC cultures, the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit (A24881; Thermo Fisher Scientific, Monza, Italy) was used, following the manufacturer’s protocol. Coverslips were mounted in DAKO fluorescence mounting medium (S3023; Agilent, Milan, Italy) and images were acquired using a Leica SP8-X confocal microscope (Leica Microsystems, Wetzlar, Germany) or a Nikon Eclipse Ti2 fluorescent microscope with a DS-Qi2 camera (Nikon, Firenze, Italy).
10
Cell Proliferation Quantification Protocol
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Cells were grown on VisiPlate-24 well black plate at a density of ∼1200 cells per well. Cells were incubated on an LPA for 72 h at different light intensities and sustained in a constant environment at 37°C and 5% . After 72 h, cells were incubated with NucBlue reagent (Thermo Fisher Scientific, catalog number: R37605) and imaged under the Nikon Eclipse Ti-E inverted fluorescence microscope. Bright spot detection was performed on each replicate under DAPI channel excitation, quantified into number of cellular objects and analyzed for cell number within MATLAB.
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