Figure 2: Influence of substrate stiffness on podosome properties.

(a) Quantification of podosomes on substrates of different stiffnesses. Human monocyte-derived macrophages were plated on fibrinogen-coated glass coverslip or polyacrylamide hydrogels coated with fibrinogen (Fg-PAs), then fixed and stained for F-actin (phalloidin-Alexa 488 nm). Means±s.e.m. of the number of podosomes per cell area are shown. Statistical analyses were performed by Dunnett’s multiple comparison test to compare the density of podosomes of macrophages plated on glass vs Fg-PA gels, and Tukey’s multiple comparison test to compare the density of podosomes in macrophages plated on Fg-PA gels of various stiffnesses. At least 100 cells from three different donors were measured per condition. ***P<0.001; *P<0.05; NS, nonsignificant. (b) Representative immunoblottings using antibodies directed against PhosphoSer19-MLC (P-MLC (S19)) on cell lysates from macrophages plated on glass coverslips coated with fibrinogen or on 25 kPa and 6.5 kPa Fg-PAs. (c) Means±s.e.m. of the PhosphoSer19-MLC levels normalized by actin immunoblot signal are shown. Statistical analyses were performed by Dunnett’s multiple comparison test (P<0.001). Lysates from three donors were analysed. (d) Quantification of podosome protrusive forces generated by macrophages plated on 30 nm-, 61 nm- or 80 nm-thick Formvar sheets corresponding to a stiffness of 6 nN μm⁻¹, 12 nN μm⁻¹ and 15 nN μm⁻¹, respectively. Means±s.e.m. of the podosome forces are shown. Statistical analyses were performed by Mann–Whitney U-test. At least 100 protrusions from three donors were measured. ***P<0.001.