Fig. 6

Reorganization of ventral actin filaments in response to soft substrates. a Airyscan images of DCs stained for actin. Shown are representative images of podosomes on stiff (left) and soft (right) substrates. The graph depicts the quantification of the length of the radiating actin filaments on stiff (n = 677 filaments) and soft (n = 609) substrates pooled from three independent experiments. Statistical analysis was performed with an unpaired two-tailed Student’s t-test. **P < 0.01. b Widefield images of DCs stained for vinculin. Shown are representative images on stiff (left) and soft (right) substrate. The graph depicts the average ± s.e.m. radial fluorescence intensity profile for vinculin on stiff (n = 948 podosomes) and soft (n = 1164) substrates pooled from three independent experiments. c Airyscan images of DCs transfected with Lifeact-GFP (magenta) and Vinculin-mCherry (green) and seeded on stiff and soft substrates. Complete cluster is shown in Supplementary Fig. 11. d Quantification of the vinculin ring diameter on stiff (n = 482) and soft (n = 627) substrates pooled from three independent experiments. Statistical analysis was performed with an unpaired two-tailed Student’s t-test. **P < 0.01 e Quantification of the FWHM of the vinculin ring on stiff (n = 515 podosomes) and soft (n = 642 podosomes) substrates. Statistical analysis was performed with an unpaired two-tailed Student’s t-test pooled from three independent experiments. *P < 0.05. f Widefield images of DCs stained for zyxin. Shown are representative images on stiff (left) and soft (right) substrate. The graph depicts the average ± s.e.m. radial fluorescence intensity profile for zyxin on stiff (n = 342 podosomes) and soft (n = 314) substrates) substrates pooled from two independent experiments. g Widefield images of DCs stained for talin. Shown are representative images on stiff (left) and soft (right) substrate. The graph depicts the average ± s.e.m. radial fluorescence intensity profile for talin on stiff (n = 376 podosomes) and soft (n = 335) substrates pooled from two independent experiments. h Airyscan images of DCs stained for actin (magenta) and vinculin (green). Shown are representative images of podosomes on glass (left), stiff (middle), and soft (right) substrate. Arrows indicate the location of the radiating actin filaments and associated vinculin. Scale bars: a–c = 1 µm, f, g = 1 µm, h = 0.5 µm. FI fluorescent intensity, AU arbitrary units. Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). A non-linear contrast enhancement (gamma correction = 0.5) was applied to all actin images (in a, c, and h) to better visualize the ventral filaments. Source data are provided as a Source Data file