Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet: Dendritic cells siphon RNA from neighboring cells through a contact dependent mechanism that does not resemble conventional means of antigen uptake (A) Outline of experiment to measure RNA and protein transfer to MutuDC1s with or without direct contact. (B) Flow cytometric analysis of SYTO62 RNA signal measured in MutuDC1s after 45 min incubation with RNA labeled COCA KCs. Results are from a single experiment. (C) Representative images and quantification of SYTO62 signal contained within MutuDC1s after keratinocyte:DC co-cultures. Mean pixel intensity of far-red channel (SYTO62) was calculated within the area occupied by GFP+ MutuDC1s on a per cell basis. Images acquired with a Nikon A1R confocal microscope using a Plan Fluor 40× Oil objective. Dots represent individual cells. Results are from a single experiment. (D) MutuDC1s (green) interacting with COCA KCs. Max projections of z stack images taken on a Nikon A1R confocal microscope using a Plan Fluor 40× Oil objective plus 10x scanner zoom. (E) Transfer of SYTO62 labeled RNA to MutuDC1s from keratinocytes relative to vehicle controls in the presence of an ATPsynthase inhibitor (1 μM Oligomycin A), an inhibitor of F-actin formation (8 μM Cytochalasin D), a macropinocytosis inhibitor (32 μM 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), and a gap junction inhibitor (5 mM 1-Heptanol). Data normalized and pooled from three experiments. Data are represented as mean ± SD.

Article Snippet: Reagents used in this study include: 8 μM Cytochalasin D (Millipore Sigma), 10 μM Oligomycin A (Selleck Chemicals), 32 μM 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Sigma Aldrich), 5 mM 1-Heptanol (Fisher Scientific), 5 mM EDTA (Fisher Scientific), 2 μM Thapsigargin (Fisher Scientific), 50 μM BAPTA-AM (Fisher Scientific), 50 μM LY294002 (Tocris), 350 nM ADH-1 (MedChem Express), 500 μg/mL Polyguanylic acid (Sigma-Aldrich), 1 mg/mL RGD peptide (Selleck Chemicals), and BLT-1 (MedChemExpress).

Techniques: Incubation, Labeling, Microscopy

Journal: Cell reports

Article Title: Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex

doi: 10.1016/j.celrep.2023.112838

Figure Lengend Snippet:

Article Snippet: After five days, we removed TMR, washed 3 times with Neurobasal, and supplemented the Neuronal media with 10μM 7-bromo-1-heptanol (Fisher Scientific, AAH5476203), which inhibits further TMR labeling.

Techniques: Ubiquitin Proteomics, Virus, Luciferase, Recombinant, Amplification, Sequencing, Software

Journal: bioRxiv

Article Title: Replication-dependent histone (Repli-Histo) labeling dissects the physical properties of euchromatin/heterochromatin in living human cells

doi: 10.1101/2024.10.20.618801

Figure Lengend Snippet: a , Multi-step photobleaching of three typical TMR-labeled replication foci. Note that the gradual decrease in fluorescent intensity indicates that each focus was labeled with multiple TMR molecules. b , The position determination accuracy of JF646-labeled nucleosomes. Distribution of nucleosome displacements from the centroids in the XY-plane in the 50-ms interval, n = 11 nucleosomes in FA-fixed HeLa cells. SDx and SDy of fitted Gaussian functions were 9.5 nm and 9.1 nm, respectively. c , MSD plots (± SD among cells) of single nucleosomes in DMSO (black, n = 24 cells) or 7BRO treated (red, n = 23 cells) HeLa cells from 0.05 to 0.5 s. Genome-wide H3.2-Halo was labeled before the 7BRO treatment. N.S.: P = 0.4 by the two-sided Kolmogorov-Smirnov test. d , Distributions of the nucleosome displacement (movement) at 50 (left), 100 (center), and 150 (right) ms. Medians of displacement are indicated in each panel. e , Schematic of the mean square displacement (MSD) calculation. f , The log-log plot of MSDs from the plot of . The plots were fitted linearly. The anomalous exponent calculated from the fitted lines is shown. g , Left: control angle distribution data from a particle with Brownian motion (also shown in ). The data was reproduced from . Center, right: motion angle distributions of nucleosomes in genome-wide-labeled living HeLa cells (n = 38 cells) and the FA-fixed control cells (n = 30 cells). Their AC values are shown at the bottom.

Article Snippet: First, asynchronous cells expressing endogenous H3.2-HaloTag were incubated with the medium supplemented with 10 µM 7-bromo-1-heptanol (7BRO, B1852; Tokyo Chemical Industry) for 60 min at 37 °C in 5% CO 2 .

Techniques: Labeling, Genome Wide, Control

Journal: bioRxiv

Article Title: Replication-dependent histone (Repli-Histo) labeling dissects the physical properties of euchromatin/heterochromatin in living human cells

doi: 10.1101/2024.10.20.618801

Figure Lengend Snippet: a , Schematic of the three-color Repli-Histo labeling for single-nucleosome imaging. Parental H3.2-Halo was labeled with green R110-direct HaloTag ligands. The remaining H3.2-Halo were then blocked with 7BRO, followed by pulse labeling with the two HaloTag ligands (TMR/JF646). b , Representative control (siControl) and RIF1-depleted (siRIF1) HeLa cells with the three-color Repli-Histo labeling. 1 st row: parental H3.2-Halo labeled with R110, identifying the S phase stage or replication timing; 2 nd and 3 rd rows: the Repli-Histo labeling patterns (TMR) and corresponding single-nucleosomes (JF646); 4 th row: the displacement of single nucleosomes as 2D heatmaps. See for the description of the labeling patterns. c , MSD plots (± SD among cells) of single nucleosomes in Class IA (red; siControl, n = 43; siRIF1, n = 27), IB (orange; siControl, n = 35; siRIF1, n = 22), II (green; siControl, n = 40; siRIF1, n = 24), and III (blue; siControl, n = 19; siRIF1, n = 23), and ambiguous patterns (grays; iControl, n = 0; siRIF1, n = 72) in siRIF1 (solid lines) or siControl (dashed lines) cells from 0.05 to 0.5 s. MSD exponents and AC values from are also shown in the brackets. d , MSD values at 0.5 s in each cell from ( c ). Black, siControl; Red, siRIF1. For siControl, ***: P = 5.6 × 10 -10 for Class IA versus IB, P = 1.9 × 10 -8 for Class IB versus II, and P = 1.6 × 10 -8 for Class II versus III by the two-sided Kolmogorov-Smirnov test. For siRIF1, **: P = 0.0012 for Class IA versus IB, P = 0.0035 for Class II versus III, and ***: P = 5.7 × 10 -10 for ambiguous patterns versus Class II. **: P = 3.2 × 10 -9 for Class IB versus II. N.S.: P = 0.70 for Class IB versus ambiguous patterns, by the two-sided Kolmogorov-Smirnov test. e , Correlation between MSD values at t = 0.5 s and R110 signals (i.e., DNA content at the time of Repli-Histo labeling). The bold “X” and bars show each stage’s mean and SD (also shown in the bottom plots). Spearman’s correlation coefficients among all cells are ρ = -0.67 for siControl and ρ = -0.57 for siRIF1.

Article Snippet: First, asynchronous cells expressing endogenous H3.2-HaloTag were incubated with the medium supplemented with 10 µM 7-bromo-1-heptanol (7BRO, B1852; Tokyo Chemical Industry) for 60 min at 37 °C in 5% CO 2 .

Techniques: Labeling, Imaging, Control

Journal: Frontiers in Physiology

Article Title: Myofibroblasts impair myocardial impulse propagation by heterocellular connexin43 gap-junctional coupling through micropores

doi: 10.3389/fphys.2024.1352911

Figure Lengend Snippet: The reagents and materials.

Article Snippet: heptanol , 17,610-32 , Nacalai Tesque, Inc..

Techniques: Cell Culture, Bioprocessing, Negative Control, Transfection