Journal: Molecular Biology of the Cell

Article Title: The fate of the primary cilium during myofibroblast transition

doi: 10.1091/mbc.E13-07-0429

Figure Lengend Snippet: Epithelial cells along a wound edge and nonepithelial progenitors lose their primary cilium as they transition into myofibroblasts. (A) Confluent LLC-PK1 layers were wounded, treated as indicated for 48 h, and stained for Ac-tub. (B) Percentage of cells with a primary cilium was determined 24 h after the indicated treatment as a function of the cell row (1–5) from the wound edge (n = 4, 25 cells/experiment for each row). (C) Human skin fibroblasts (HSFs; left) and 10T1/2 cells (right) were serum starved or treated with TGFβ (5 ng/ml) for 72 h, followed by Western blotting for the indicated proteins. (D) The same two cell types were treated as in C and then stained for Ac-tub. Nuclei were visualized with 4′,6-diamidino-2-phenylindole. (E) 10T1/2 cells were treated as in C and then processed for scanning electron microscopy and visualized using magnification 2500× (left) and 15,000× (right). Right, enlarged area indicated by boxes on the left. Bars, 10 and 1 μm for upper and lower panels, respectively.

Article Snippet: Fixed samples were blocked using 3% bovine serum albumin in PBS (1 h to overnight), followed by primary antibody incubation for 1 h. Cells were then washed with PBS and incubated with fluorescently labeled secondary antibodies for 1 h with the addition of 4′,6-diamidino-2-phenylindole (Lonza, Basel, Switzerland) for nuclear labeling.

Techniques: Staining, Western Blot, Electron Microscopy