Journal:

Article Title: Erk and PI-3 Kinase Are Necessary for Collagen Binding and Actin Reorganization in Corneal Epithelia

doi:

Figure Lengend Snippet: Epithelia treated with MAP kinase inhibitor, PD98059. Confocal images through the ACM optical plane (Fig. 1E, arrowhead) of whole epithelial tissue stained with FITC-phalloidin after incubation with PD98059 (50 and 100 μM; B, C) and stimulation with COL for 2 hours compared with control tissue (A). The lowest dose of PD98059 had little effect on ECM-stimulated ACM reformation; however, at the middle (B, arrowheads) and highest (C, arrowheads) doses, the blebs were more prominent. Western blot of activated erk-1 and -2 (D) showed a decrease in activity of these MAP kinase proteins from cell extracts that were treated with PD98059 in a dose-dependent manner compared with untreated epithelia (0) or control cell extracts (A431). The blot was reprobed with β-tubulin to demonstrate that the gel was loaded equally. Densitometry analyses (E) of the anti active erk-1 and -2 Western blot (D). β-Tubulin was used to obtain corrected densities (OD units) of the two proteins, erk-1 and -2. Both erk-1 and -2 decreased in a dose-dependent manner compared with uninhibited control tissue (0). Scale bar, 10 μm.

Article Snippet: A densitometry computer program from the microscope manufacturer (Zeiss) was used to determine the average intensity (range = 0–255) of the pixels that intersected lines through the center of the tissue and at the basal cell surface ( , yellow lines).

Techniques: Staining, Incubation, Control, Western Blot, Activity Assay

Journal:

Article Title: Erk and PI-3 Kinase Are Necessary for Collagen Binding and Actin Reorganization in Corneal Epithelia

doi:

Figure Lengend Snippet: Epithelia treated with the PI-3 kinase inhibitor LY294002. Single confocal images taken through the ACM optical plane of FITC-phalloidin–stained epithelial tissue after incubation with PI-3 kinase inhibitor LY294002 (50 and 100 nM) and stimulated with COL in the presence of inhibitor for 2 hours (B, C) compared with control COL-treated epithelia (A). Tissue treated with 50 nM LY294002 (B, arrowheads), had actin filaments in the basal region of the tissue; however, they did not align from cell to cell as seen in the control tissue (A, arrowheads). In contrast, tissue treated with 100 nM LY294002 (C, arrowheads) had a complete loss of actin filament bundles in the ACM region. Western blot of the 85-kDa PI-3 kinase subunit (D) showed a decrease in the protein from cell extracts that were treated with LY294002 compared with untreated epithelia (0) or control cell extracts (A431). Densitometry analysis (E) of the PI-3 kinase Western blot (D) corrected for loading with β-tubulin. The amount of 85-kDa was reduced approximately 25% in all treated doses compared with untreated tissue (0). The protein levels did not decrease with increasing doses of LY294002. Scale bar, 10 μm.

Article Snippet: A densitometry computer program from the microscope manufacturer (Zeiss) was used to determine the average intensity (range = 0–255) of the pixels that intersected lines through the center of the tissue and at the basal cell surface ( , yellow lines).

Techniques: Staining, Incubation, Control, Western Blot

Journal:

Article Title: Erk and PI-3 Kinase Are Necessary for Collagen Binding and Actin Reorganization in Corneal Epithelia

doi:

Figure Lengend Snippet: COL binding and F-actin accumulation in the presence of MAP kinase or PI-3 kinase inhibitors. Epithelia were cultured in the presence and absence of either MAP kinase (100 μM PD98059) or PI-3 kinase inhibitor (100 nM LY294002) and stimulated with FITC-labeled COL for 30 or 60 minutes. A complete z-series of confocal images were projected into one image. Then an xz image was produced from the projected three-dimensional stack (A). These final images contained all the image pixels from the complete set of z-series images. Average intensities (scale range, 0–255) of bound COL (green) and F-actin (red) were determined using a line drawn through the center of the basal cells (upper line in A, PD98059 tissue) or at the base of the epithelia (lower line in A, PD98059-treated tissue). Densitometry software was used to determine average intensities of bound COL (green channel intensity levels, n = 4 epithelia per group) cellular F-actin (red channel intensity levels, n = 4 epitheliaper group). In the basal compartment, there was a decrease in both bound COL (B, *P ≤ 0.05) and F-actin (C) in PI-3 kinase– and MAP kinase–inhibited epithelia compared with control epithelia. The actin accumulation in the center of the basal cells also demonstrated a marked difference between groups at 30 minutes (C, *P < 0.05). However, by 60 minutes, there was no difference between groups (C). Collagen-coated polystyrene beads were used to further quantitate the differences in collagen binding (D). The tissues were incubated basal side up and treated with inhibitors, and the collagen-coated beads were added to the medium for 30 minutes. The tissues were placed in a black 96-well dish (1 epithelium per well; n = 5–10 epithelia per treatment group) and analyzed with a fluorometer. The intensity of each well was analyzed with both FITC and rhodamine filter sets. The mean intensity and SDs were calculated for each group. Tissue treated with 100 nM LY294002 had 43% intensity compared with controls, whereas tissues treated with 1 μM LY294002 had less than 10% intensity. Tissues treated with 100 μM PD98059 had 14% of control intensity.

Article Snippet: A densitometry computer program from the microscope manufacturer (Zeiss) was used to determine the average intensity (range = 0–255) of the pixels that intersected lines through the center of the tissue and at the basal cell surface ( , yellow lines).

Techniques: Binding Assay, Cell Culture, Labeling, Produced, Software, Control, Incubation