Figure 4: Cell spreading and cellular F-actin content in CRN7 KO fibroblasts.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) Actin cytoskeleton stained with TRITC-phalloidin (red) and focal adhesion stained with vinculin (green) after 60 min of spreading on FN-coated wells analysed by fluorescence microscopy. Scale bar = 10 μm. (b) Statistical analysis of (a). Line graph shows the increase in projected cell area at different time points (15, 30, 60 min) measured using polygon tool of LAS AF Lite (n = 200 cells each, per time point, 2 independent experiments; *P < 0.05). (c) Representative images of KO and rescue fibroblasts (glowing green) stained for F-actin only (red) as in (a). Scale bar = 25 μm. (d) Fibroblasts quantified for increase in projected cellular area as before (15, 30, 60 min) and represented as line chart (n = 40 cells each, each time point, 2 independent experiments; *P < 0.05). (e) Cells fixed and stained as in (a) and confocal z-stack images (step size 10) taken. Blue (255), higher actin intensity in the selected channel; yellow to green, decreasing intensity. Scale bar = 25 μm. (f) Fluorescence intensity measurement done by a z-stack analysis using confocal microscopy (n = 25 view fields, 3–4 cells per view field, each cell type; **P < 0.01). (g) Representative images of fully spread KO and rescue cells (green = GFP) stained for F-actin as in (e). Scale bar = 25 μm. (h) Fluorescence intensity measurement done as in (f ) (n = 40 cells each, 2 independent experiments; **P < 0.01). AU, arbitrary units. Data shown as mean ± SD.
