A 2D and 3D cell culture protocol to study O-GlcNAc in sphingosine-1-phosphate mediated fibroblast contraction

STAR Protoc. 2022 Jan 20;3(1):101113. doi: 10.1016/j.xpro.2021.101113. eCollection 2022 Mar 18.

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc's role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures. We then describe 2D contraction assay and 3D collagen gel contraction assay to analyze the effect of the modification on sphingosine-1-phosphate signaling for contraction. For complete details on the use and execution of this protocol, please refer to Pedowitz et al. (2021).

Keywords: Biophysics; Cell Biology; Cell culture; Cell-based Assays; Microscopy; Molecular/Chemical Probes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Animals
  • Cell Culture Techniques
  • Fibroblasts / metabolism
  • Humans
  • Lysophospholipids / metabolism*
  • Mice
  • NIH 3T3 Cells
  • Protein Processing, Post-Translational
  • Signal Transduction
  • Sphingosine / analogs & derivatives*
  • Sphingosine / metabolism

Substances

  • Lysophospholipids
  • sphingosine 1-phosphate
  • Sphingosine
  • Acetylglucosamine