Fig. 4

Fibronectin EDA determines topological and mechanical properties of myofibroblastic matrices. A CAF-derived 3D ECMs. Indicated CAFs were seeded on coverslips and allowed to produce ECM for 6 days. Cell cultures were then fixed and analyzed by immunofluorescence (IF) with an anti-fibronectin (red), anti-fibronectin EDA (green) and DAPI (white). B Quantification of the oriented CAF nuclei within 3D ECMs. Orientation angles of the DAPI-stained nuclei were calculated using the ImageJ analysis particles tool. Percentage of nuclei orientated toward the most frequent angle (up to 21° deviation) is shown. C Relative RNA amount of EDA+ fibronectin isoforms in genetically modified MEF lines. RNA from control, EDA– and EDA+ MEFs treated with 5 ng/mL of TGFβ₁ for 3 h was retrotranscribed and amplified using primers flanking exon 33 of Fn1 (as described in Fig. 1) and visualized by DNA-electrophoresis. D Relative protein amount of EDA+ fibronectin isoforms in genetically modified MEF lines. Indicated MEFs treated as in C were lysed in SDS buffer, and the levels of the indicated proteins were analyzed by Western blot. E Fibronectin fibers in 3D ECMs. Indicated MEFs seeded on coverslips were allowed to produce extracellular matrix for 6 days in the presence or absence of 5 ng/mL TGFβ₁. Cell cultures were then fixed and analyzed by IF with an anti-fibronectin (green) and DAPI. Confocal and STED microscopy were used to obtain images. F Quantification of fibronectin fiber alignment in 3D ECMs. The fiber angles were calculated using the ImageJ plugin OrientationJ. The percentage of fibers aligned toward the same direction (up to 21° deviation from the mode) is shown. G Quantification of fibronectin fiber alignment index through TWOMBLI. Fibronectin fiber images obtained as in E were analyzed using the ImageJ macro TWOMBLI. All obtained data are plotted, showing all individual measurements, mean and SEM. H Quantification of fibronectin fiber parameters through TWOMBLI. The indicated parameters were analyzed from images used in E. Arbitrary units provided by the plugin are expressed relative to wild-type MEFs. I Visualization of collagen deposition from in vivo–like extracellular matrices. 3D ECMs were produced as in C fixed with 4% PFA and stained with Masson's trichrome. J, Quantification of the stiffness of in vivo–like extracellular matrices. 3D ECMs generated as in E were decellularized, and the elastic modulus was calculated from atomic force-curve measurements