Supplementary Figure 2: 3D rendering of endothelial podosome rosettes in RipTag2 tumor and ischemic vessels.
From: Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis
(a) Schematic representation of endothelial tumoral rosettes detection. Endothelial cells (EC) are delimited by red line while vBM is colored in magenta, in green there is the schematization of endothelial tumoral rosettes visualized as shown in Fig. 2A. Yellow arrows indicate the rosettes. (b) 3D reconstruction of a representative endothelial rosette in RipTag2 tumors. Isosurface of vBM – detected as laminin staining – was coloured in red, endothelial rosettes – F-actin/cortactin colocalization – in yellow and nuclear staining in blue. Scale bar, 5 μm. (c) Confocal imaging stacks of representative vessels in hindlimb ischemia experiment on gastrocnemius muscles. Xyz-section of immunostaining for primary Abs as indicated. Vessels are delimited by white dotted lines; white arrows indicate podosome-rosettes. Scale bar, 10 μm. (d) vBM quantification in different regions of tumor vessels in RipTag2 tumors. vBM was detected as laminin staining. vBM volumes were subdivided in 1000-μm3-volumes. Therefore, the subvolumes of vBM were classified in vBM volumes without endothelial rosettes (no rosette vBM volumes) and vBM volumes with endothelial rosettes (rosette vBM volumes). Normalized mean ± SEM of n = 420 subvolumes of vBM from 5 fields per mouse for a total of 3 mice per treatment group. (∗∗∗, P < 0.001 versus no rosette vBM volumes). Statistical significance was calculated using unpaired nonparametric Mann-Whitney test. (e) 3D reconstruction of in situ zymography in a representative endothelial rosette of RipTag2 tumors. After deconvolution, isosurface of vBM – detected as laminin staining – was coloured in magenta, F-actin in red and Gelatin-DQ – indication of gelatin degradation – in green. Scale bar, 10 μm. (f) In situ zymography in lung tumors from patients. Staining for primary Abs as indicated and Gelatin-DQ (dye-quenched), i.e. degradated gelatin. Vessel is delimited by white dotted lines; white arrows indicate podosome-rosettes. Scale bar, 10 μm.
