Journal: The Journal of Biological Chemistry

Article Title: Using CRISPR-Cas9/phosphoproteomics to identify substrates of calcium/calmodulin-dependent kinase 2δ

doi: 10.1016/j.jbc.2023.105371

Figure Lengend Snippet: Tight junction, distribution of actin cytoskeleton, and nuclear morphology in the presence of dDAVP. A and B , confocal images of Camk2d intact and KO cells labeled with antibody recognizing Zonula Occludens 1 (ZO-1) protein. Epithelial polarization and the cell cross sectional area was unaffected by Camk2d deletion. The ZO-1 intensity and cross sectional area were calculated from the cell shown entire image field using ImageJ. Nuclear counts per unit area were used to estimate cell density. Maximum intensity Z-projection of ZO-1 was divided by the number of cells. Three randomly chosen areas from three intact and five KO cell lines were used for analysis. C and D , nuclei volume and surface areas in intact and KO cells. Nuclei volume and nuclear surface area were calculated with Imaris software. The calculation showed no significant changes in Camk2d KO cells compared to the intact cells. Nuclei volume and surface area were calculated using the Surfaces tool of Imaris. A total of 240 nuclei from intact cells (three random area per clone, three clones) and total 468 nuclei from KO cells (three random area per clone, five clones) were analyzed. E and F , confocal images of Camk2d intact and KO cells labeled with phalloidin. Subcellular distribution of F-actin was not demonstrably changed with Camk2d deletion. The sum of phalloidin intensities and the sum of DAPI intensities were obtained from Imaris. The nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI). Morphometry was carried out in three randomly selected areas for both intact and KO clones. Confocal microscope magnification 63×. Scale bar without a number represents 5 μm. Three randomly chosen areas from three intact and five KO cell lines were used for analysis. p values are from unpaired t-tests throughout. CAMK2D, CAMK2δ; dDAVP, 1-desamino-8-D-arginine-vasopressin.

Article Snippet: The sum of phalloidin intensities and the sum of DAPI intensities were obtained from Imaris.

Techniques: Labeling, Software, Clone Assay, Staining, Microscopy

Journal: BMC neuroscience

Article Title: NPT100-18A rescues mitochondrial oxidative stress and neuronal degeneration in human iPSC-based Parkinson's model.

doi: 10.1186/s12868-025-00926-y

Figure Lengend Snippet: Fig. 1 Increased oxidative stress and mitochondrial dysfunction in patient-derived mDANs. (A) Schematic of experimental design. Fibroblasts from PD patients carrying a monoallelic SNCA locus duplication and unaffected control individuals were obtained through punch biopsy of the skin. Fibroblasts were reprogrammed into human iPSCs. Human iPSCs were first differentiated into NPCs through dual SMAD inhibition and activation of canonical Wnt signaling and then into mDANs using a FGF8b-based protocol. mDANs were treated with NPT100-18A during the entire differentiation and maturation periods. (B-D) Relative levels depicted as fold changes from the mean of control cell line C1 ± SD of n wells from N independent differentiations. Repli cates belonging to the same differentiation are shown in the same color. (B) CellRox Green fluorescence intensities (FIs) were assessed in living mDANs. CellRox and DAPI FIs were measured at 80 sites per well in 96-well plates using a CLARIOstar plate reader. CellRox FIs were normalized to respective DAPI FIs. Dots representing single well means (n = 15, N = 4). Right panels show representative microscopy images of CellRox and DAPI fluorescence signals. (C) Significantly reduced relative ATP levels measured in patient-derived compared to control mDANs. Relative ATP levels were assessed in mDAN lysates using a luciferase-based assay and a LUMIstar Omega plate reader. Values for single wells were normalized to the frequency of viable neurons (determined with a viability staining in mDANs cultured in parallel under the same conditions). Dots represent single wells (n = 9, N = 3). (D) MitoSOX Red FIs were measured in living mDANs analogously to (B). Dots represent single well means (n = 18, N = 4). Right panels show representative microscopy images of MitoSOX and DAPI fluorescence signals; for better visualization of neurons, β3-tubulin (TUBB3) was additionally immunofluorescently labelled. (B-D) Student’s t-test, *P < 0.05, ** P < 0.01, ***P < 0.001. Scale bar 50 μm in (B) and 20 μm in (D)

Article Snippet: CellROX, MitoSOX, and DAPI fluorescence intensities (FIs) were measured on a CLARIOstar Plus microplate reader (BMG Labtech) using the following excitation/emission wavelengths: CellROX – 500 − 20/530 − 25 nm; MitoSOX – 510 − 15/580 − 20 nm; DAPI – 360 − 20/460 − 30 nm.

Techniques: Derivative Assay, Control, Inhibition, Activation Assay, Fluorescence, Microscopy, Luciferase, Staining, Cell Culture

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Binding of human LEDGF/p75 and HIV-1 IN to chromosomes spreads. Either recombinant purified LEDGF/p75 or HIV-1 IN (4nM) were incubated alone (respectively A and B ) or together ( C ) with chromosomes spreads under the conditions described in materials and methods section. Monitoring of their interaction with the chromatin was performed by immunofluorescence with the corresponding antibody coupled to ALEXA 594 (green signal) and DAPI staining (red signal). The intensity of total IF signal has been quantified in each conditions using ImageJ software and the IN binding without or with LEDGF/p75 were reported in ( D ) as mean from the quantification of 7–11 chromosomes in each condition ± standard deviation (SD), P -value was calculated by student test. Scale bar = 10 μM.

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Binding Assay, Recombinant, Purification, Incubation, Immunofluorescence, Staining, Software, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Correlation analysis between the protein distributions and chromatin features. Distributions of IN, LEDGF/p75 and IN-LEDGF/p75 were compared to DAPI staining and histones distribution. The mean and standard deviation of the pearson correlation calculation (>0 for a correlation, =0 for no correlation and < 0 for a negative correlation) were performed between all paired protein distribution and DAPI/histone curves. The p-value (Wilcoxon rank sum test) at each position and the percentage of pairs with P < 0.01 was also calculated and were reported as *** if > 0.80 and ** if > 0.40).

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Staining, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Analysis of the chromatin binding property of HIV-1 intasome. HIV-1 intasome was assembled using IN, LEDGF/p75 and the viral short DNA sequence corresponding to the viral end tagged with FITC. The intasome was purified by size exclusion chromatohraphy (see S8). After verification of their functionality in in vitro concerted integration assays the intasome (4nM) were incubated with chromosomes spreads. Interaction profile was determine by FITC-epifluorescence acquisitions ( A ). The distribution profile onto chromosome 1 was compared determined as reported in materials and methods section (an example of chromosome 1 is reported in ( B ), see the profile of chromosome 2 and 3 in S9). The data are reported as means from the quantification of eight to nine chromosomes ± standard deviation (SD). Scale bar = 10 μM. Correlation between the distributions of the intasome and the distribution of IN alone, LEDGF/p75 alone or IN-LEDGF/p75 complex ( C ) as well as DAPI staining, H3/H4, H3K36me3, K3K27me3 and H3K27ac ( D ) was compared. The mean and standard deviation of the Pearson correlation calculation (>0 for a positive correlation, =0 for no correlation and <0 for negative correlation) were performed between all paired protein distribution and DAPI/histone curves. The P -value (Wilcoxon rank sum test) at each position and the percentage of pairs with P < 0.01 was also calculated and were reported as *** if > 0.80 and ** if > 0.40).

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Binding Assay, Sequencing, Purification, In Vitro, Incubation, Standard Deviation, Staining