Journal: Cell Adhesion & Migration

Article Title: Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

doi: 10.4161/cam.4.4.12661

Figure Lengend Snippet: Characterization of A431 cells expressing GFP-E-cadherin, PAGFP-E-cadherin and PAGFP-Farn2Palm. Expression and cellular localization of GFP-E-cadherin, PAGFP-E-cadherin and PAGFP-Farn2Palm in stable pools of A431 were confirmed by immunoblot of E-cadherin and GFP (A) and immunofluorescence of GFP (B). (C) Montage showing cellular localization of GFP-E-cadherin at different levels in the z-axis of the cells. The asterisk (*) indicates the focal plane chosen for E-cadherin dynamics analyses. Blue, DAPI staining. Scale bars, 20 µm.

Article Snippet: A431 skin epidermoid carcinoma cells were obtained from the American Type Tissue Collection (LGC Promochem) and cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Invitrogen).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Cell Adhesion & Migration

Article Title: Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

doi: 10.4161/cam.4.4.12661

Figure Lengend Snippet: E-cadherin is mainly retained at the plasma membrane of confluent monolayers of A431 cells. (A) Segmentation of fluorescence signal using ImageJ and corresponding still images from photoactivation experiments at activation and t1/2 time points for PAGFP-E-cadherin and PAGFP-Farn2Palm used for membrane retention analyses. (B) Quantification of the proportion of fluorescence intensity that is retained in the plasma membrane at t1/2 following activation of PAGFP-E-cadherin and PAGFP-Farn2Palm. (t test, *p = 0.003). Values represent the mean from at least 25 cells. Error bars, s.e.m.

Article Snippet: A431 skin epidermoid carcinoma cells were obtained from the American Type Tissue Collection (LGC Promochem) and cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Invitrogen).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Activation Assay

Journal: Cell Adhesion & Migration

Article Title: Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

doi: 10.4161/cam.4.4.12661

Figure Lengend Snippet: Deregulation of intracellular trafficking specifically alters the rate of movement of E-cadherin at the plasma membrane. (A) Quantification of biotinylated E-cadherin internalization over 10 min in A431 cells or A431 cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 30 min). (B) Fluorescence recovery curves following photobleaching of GFP-Farn2Palm (left part) and GFP-E-cadherin (right part) in control cells (blue) or cells treated with dynasore (red) or bafilomycin A1 (green), (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). (C) Quantification of t1/2 following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1. (D) Quantification of the speed of lateral movement of PAGFP-E-cadherin within the membrane in cells treated with bafilomycin A1. Values represent the mean from at least 25 cells. Error bars, s.e.m.

Article Snippet: A431 skin epidermoid carcinoma cells were obtained from the American Type Tissue Collection (LGC Promochem) and cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Invitrogen).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Control

Journal: Cell Adhesion & Migration

Article Title: Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

doi: 10.4161/cam.4.4.12661

Figure Lengend Snippet: Inhibition of endocytosis strengthens cell-cell adhesion. (A) Representative images of fragments detached from a dispase-treated monolayer of control or dynasore (80 µM, 30 min) pre-treated A431 cells after mechanical stress. (B) Quantification of the number of single cells that disaggregate from a dispase-treated monolayer after mechanical stress. (C) Quantification of the number of single cells that disaggregate after mechanical stress from a dispase-treated monolayer of A431 or A431 GFP-E-cadherin cells, transfected with either control or E-cadherin siRNA. (D) Still images obtained from photobleaching experiments showing GFP-E-cadherin localization in A431 cells untreated or treated with dynasore (80 µM, 30 min). Scale bars, 20 µm. (E) Immunoblot showing surface and total levels of GFP- and endogenous E-cadherin (150 kDa and 120 kDa, respectively) in A431 cells untreated or treated with dynasore (80 µM, 30 min). (B and C) Values represent the mean from at least three independent experiments. Error bars, s.e.m.

Article Snippet: A431 skin epidermoid carcinoma cells were obtained from the American Type Tissue Collection (LGC Promochem) and cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Invitrogen).

Techniques: Inhibition, Control, Transfection, Western Blot