Figure 5
Podoplanin mediates invadopodia stabilisation. (a–c) HN5 control (Sc) and podoplanin-depleted (PDPN sh3 and sh4) cells expressing Lifeact-GFP were cultured on 0.5% glutaraldehyde-crosslinked gelatin for 3 h. Live-cell imaging was performed and analysed to calculate invadopodia lifetime (a, b) and total number of invadopodia per cell (c). N⩾90 invadopodia per condition from 10 cells over three independent experiments. Error bars represent s.e.m. An animate sequence of these data is shown in Supplementary Movie S3. (d) Invadopodia disassembly rates in control and podoplanin-depleted cells. Cells were grown on crosslinked unlabelled gelatin overnight. Invadopodia disassembly was induced by treatment with 10mM methil-β-cyclodextrin (MβCD) for 20 min. Cells were fixed at the indicated time points, double stained for actin and cortactin and the number of cells with invadopodia was determined. (e) Invadopodia reassembly rates were determined after washout of MβCD and incubation with normal medium. Rates of invadopodia disassembly and reassembly were calculated as the percentage of cells forming invadopodia on each time point. Values were normalised to t=0 min (disassembly) or to t=20 min after MβCD treatment (reassembly). The results shown are the means±s.e.m. of n⩾200 cells for each condition and time point over three independent experiments. Bars=20 μm. ***P<0.0005; * P<0.05; NS=not significant.
