mouse fibroblast nih3t3  (ATCC)

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: STAR Protocols

Article Title: Protocol to identify mechanosensitive nuclear proteins using tunable actomyosin contractility and proximity biotinylation in mammalian cells

doi: 10.1016/j.xpro.2025.104288

Figure Lengend Snippet: Mechanosensitive responses of PTBP1 under various mechanical conditions (A) Volcano plot demonstrating significant nuclear enrichment of PTBP1 following tetracycline treatment (FDR< 0.01). Reprinted data with permission from Tseng et al. (B) Western blot analysis of PTBP1 nuclear enrichment, with YAP as a positive control. (C) Diagram shows the mechanical conditions used in this protocol to verify PTBP1 expression. Immunofluorescence microscopy confirming the nuclear accumulation of PTBP1 induced by high mechanical tension including low cell density (D, scale bar: 20 μm), stiff substrate (E, scale bar: 10 μm), and mechanical stretching (F, scale bar: 50 μm). (G) Immunofluorescence images showing PTBP1 nuclear reduction in ML-7 treated NIH3T3. Bar represents 10 μm.

Article Snippet: NIH3T3 , ATCC , #CRL-1658; RRID: CVCL_0594.

Techniques: Western Blot, Positive Control, Expressing, Immunofluorescence, Microscopy

Journal: Bioactive Materials

Article Title: Hydrogel delivering antifibrotic agent and nano-sonosensitizer enhances efficacy of sonodynamic therapy in osteosarcoma treatment

doi: 10.1016/j.bioactmat.2025.10.001

Figure Lengend Snippet: SIS3 reprograms CAFs (a) Schematic representation depicting the in vitro activation of NIH3T3 cells into CAFs using an OS-conditioned medium. (b) Western blotting is used to assess the expression levels of CAFs activation markers α-SMA and FAP. (c) Immunofluorescence (IF) staining is used to visualize the expression of CAFs activation markers α-SMA and FAP (scale bar: 25 μm). (d) IF staining is used to detect the expression of α-SMA and FAP in NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 25 μm). (e) Statistical analysis of relative fluorescence intensity (FI), presented as mean ± SD (n = 3). (f) Schematic illustration of the TGF-β/SMAD3 signaling pathway. (g) Western blotting is used to evaluate the activation status of the TGF-β/SMAD3 signaling pathway in NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 and the statistical analysis of relative SMAD and p-SMAD expression, presented as mean ± SD (n = 3). (h) Representative images from collagen gel contraction assays on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 and the statistical analysis of relative contraction degree, presented as mean ± SD (n = 3). (i) Representative images from wound healing assays conducted on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 200 μm) and the statistical analysis of relative wound healing degree, presented as mean ± SD (n = 3). (j) Representative images from transwell migration assays performed on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 100 μm) and the statistical analysis of the relative number of migrated cells, presented as mean ± SD (n = 3).

Article Snippet: The human OS cell line 143B, the mouse OS cell line K7M2, the mouse embryonic fibroblasts NIH3T3, and the human umbilical vein endothelial cells HUVEC were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: In Vitro, Activation Assay, Western Blot, Expressing, Immunofluorescence, Staining, Cell Culture, Fluorescence, Migration