Figure 3: Myosin IIA-mediated contractility is not essential for podosome ring integrity.

Immunolocalization of actin (red) with either (in green) (a) vinculin, (b) talin, (c) zyxin and (d) paxillin in untreated control cells (upper row) or cells treated for 30 min with 20 μM blebb (lower row). Actin was stained by Texas Red phalloidin, whereas adaptor proteins were labelled by specific monoclonal antibodies and subsequently secondary antibodies conjugated to Alexa488. Merged images are shown on the right. Images are representative of multiple cells from at least three similar experiments. Scale bar, 2 μm. (e) Levels of vinculin, zyxin, talin and paxillin were measured in at least 800 podosomes per condition. Data are normalized to the average intensity and blebb-treated levels are expressed relative to control levels. Shown are the median (middle line) and the interquartile range (upper and lower whiskers). Statistical analyses were performed by two-tailed Student’s t-test. ***P<0.001. (f,g) DCs were transfected with vinculin-GFP or zyxin-GFP. Shown are the vinculin and zyxin level variations over time of randomly selected podosomes in (f) untreated control cells or (g) cells treated for 30 min with 20 μM Y27632. Data are normalized to the average intensity.