Figure 3. Pharmacological antagonism of eIF4E blocks TGF-β1 mediated actin reorganization and motility.

Primary rat AT2 cells were seeded onto type 1 collagen coated filters at a density of 5 × 10⁵ cells/cm². After 2 days, cells were treated with TGF-β1 (2.5 ng/ml) with or without 4Ei-1 (500 μM) with medium replenished every other day until day 14 when they were fixed with 4% paraformaldehyde or harvested for protein. (a) Shown are immunofluorescence images with anti-α-SMA antibody (green) and propidium iodide (red) to visualize nuclei; upper panel at x20 (Bar = 50 μm), lower panel at x40 (Bar = 15 μm). (b) Shown is an immunoblot (left panel) probed for α-SMA with GAPDH as the loading control. Immunoblot shown is representative of 4 independent experiments. The mean quantitation values for all 4 replicates are shown (right panels). *indicates P < 0.05 for the comparison of vehicle versus TGF-β1 and TGF-β1 + 4Ei-1 versus TGF-β1. (c) RLE-6TN cells were seeded onto cell culture inserts and treated with vehicle or TGF-β1 (2.5 ng/ml) ± 4Ei-1 (100 μM) for 72 h. Shown for each condition is the migration index [migration of cells treated with TGF-β1 or (TGF-β1 + 4Ei-1)]/[migration of cells treated with vehicle control]. *indicates P < 0.05.