nih3t3  (ATCC)

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Exploration of optogenetic clustering properties of CRY2. (A) Top panels, time-lapse images of a NIH3T3 cell expressing CRY2high–mCherry activated with a 488-nm microscope laser starting at time t =0 (blue vertical arrow, 1.5 s pulses every 10 s). Scale bars: 5 µm. Bottom panel, coefficient of variation (CV) of fluorescence intensity calculated as the ratio between the nuclear intensity standard deviation and the nuclear intensity mean, presented as relative to the CV at time t =0. Data points corresponding to the images are marked in red. (B) Top panel, protein sequence of the C-terminus of the CRY2 PHR domain and part of the artificial linker used for C-terminal fusions for wild-type CRY2 (CRY2wt) and for CRY2 mutants. The newly generated variant CRY2hiclu is marked in bold. Mutations relative to the CRY2wt sequence are highlighted in gray. Bottom panel, images of NIH3T3 cells expressing CRY2 mutants fused to mCherry, illuminated with 1 s blue light pulses every 10 s for 15 min, and then fixed. The nucleus is delimited with a yellow line. Scale bars: 5 µm. (C) CV calculated from images obtained from NIH3T3 cells expressing CRY2 variants fused to mCherry, illuminated with pulsed blue light for 15 min, and then fixed, plotted as a function of mCherry nuclear intensity. ∼25 cells were analyzed per sample (each dot represents one cell). Continuous lines represent simple logistic fits. (D) Time-lapse images of a NIH3T3 cell expressing CRY2hiclu–mCherry activated once with the 488-nm microscope laser for 15 s at time t =0 (marked with a blue arrow). Scale bars: 5 µm. (E) Mean ( n =25) CV calculated from time-lapse images obtained from NIH3T3 cells expressing CRY2olig-mCherry, illuminated with blue light at time t =0, and then kept without blue light. The clustering ( t c ) and declustering times ( t d ) were determined from individual kinetic curves. (F) t c (top panel) and t d (bottom panel) represented as a function of mCherry nuclear intensity. ∼25–40 cells were analyzed per sample (each dot represents one cell). Continuous lines represent simple exponential (clustering) and linear (declustering) fits.

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Microscopy, Fluorescence, Standard Deviation, Sequencing, Generated, Variant Assay

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: c-MYC is an aggregation-prone, amyloidogenic protein

doi: 10.64898/2026.03.12.711438

Figure Lengend Snippet: (A) Measurements of the transcriptional activities of HA-MYC WT and HA-MYC ΔC in HeLa cells using the dual MYC reporter system (mean ± SD, n=3 independent experiments, One-way ANOVA). (B) Quantitation of apoptosis induced by transient expression of HA-MYC WT or HA-MYC ΔC in serum-starved NIH3T3 cells by flow cytometry using anti-cleaved caspase 3 Abs (mean ± SD, n=3 independent experiments, One-way ANOVA). (C) Detection of HA-MYC expression in serum-starved NIH3T3 cells by immunoblotting (representative images of four independent experiments). (E) Quantitation of apoptosis induced by transient expression of HA-MYC ΔC and its deletion mutants in serum-starved NIH3T3 cells by flow cytometry using anti-cleaved caspase 3 Abs. The results are normalized by their expression levels (mean ± SD, n=4 independent experiments, One-way ANOVA).

Article Snippet: HeLa (female) cells and NIH3T3 (male) cells were purchased from ATCC.

Techniques: Quantitation Assay, Expressing, Flow Cytometry, Western Blot