Fig 2: KLK12 modulates fibronectin matrix assembly by endothelial cells. The impact of KLK12 on FN matrix assembly by human ECs was analyzed after treatment with 10 nM KLK12 for 4 h or 24 h in basal medium containing 500 nM FN or FN-EDB. (A) Immunofluorescence analysis. After treatment, cells and matrix were fixed, FN fibrils were immunodetected (monoclonal anti-FN antibody IST-2, green channel), and DNA was stained using Hoechst reagent (blue channel). Scale bar = 50 µm. (B) Western blot. After treatment, the DOC-insoluble protein fraction and conditioned media were harvested and analyzed by Western blotting. Upper panel: Detection of FN fibrils in the DOC-insoluble protein fraction using the IST-2 anti-FN monoclonal antibody. Lower panel: Detection of soluble FN in conditioned media using the FN9-1 antibody directed against the amino-terminal domains of FN. The sizes of the protein markers (in kDa) are indicated on the left.

Fig 3: Fibronectin and KLK12 are both expressed in the vascular endothelium. Immunofluorescent staining of capillaries formed after 9 days of fibroblast-EC co-culture. Top panel: Staining of KLK12 (green), PECAM/CD31 (red), and nuclei (blue). Bottom panel: Staining of FN (green), PECAM/CD31 (red), and nuclei (blue). Colocalization of KLK12 or FN with capillaries (CD31/PECAM staining) appears in yellow. Scale bars correspond to 50 µm for 20x images and 30 µm for 40x images.

Fig 4: KLK12 neutralization modifies fibronectin matrix assembly by endothelial cells and fibroblasts. (A) Description of the protocol used to generate pAbFN-KLK12, an antibody that recognizes the KLK12 cleavage site. (B) Test of pAbFN-KLK12′s specificity on KLK12-generated FN fragments. KLK12-generated proteolytic fragments of FN and FN-EDB were prepared as in Fig. 2A and then analyzed in Western blot. (C and D) The impact of KLK12 neutralization on FN matrix assembly by ECs (C) and fibroblasts (D), using a neutralizing antibody that targeted the KLK12 cleavage site on FN (pAbFN-KLK12). Human ECs were treated with 10 nM KLK12 for 24 h in basal medium with or without FN (500 nM) and pAbFN-KLK12 or the isotype control (500 nM). Fibroblasts were treated with 10 nM KLK12 for 24 h in basal medium with or without pAbFN-KLK12 (50 nM and 500 nM). After treatment, cells and matrix were fixed, the FN fibrils were immunodetected (monoclonal anti-FN antibody IST-2, green channel) and DNA was stained using Hoechst reagent (blue channel). Scale bar = 200 µm.

Fig 5: KLK12 exerts proteolytic activity against fibronectin isoforms. (A) Proteolysis of matrix proteins by KLK12, analyzed with SDS-PAGE and Coomassie blue staining (E:S molar ratio: 1:5, 4 h at 37 °C). (B) Characterization of FN proteolysis by KLK12, analyzed as in A. Left panel: Dose-dependent proteolysis of FN by KLK12 with an E:S molar ratio ranging from 1:5 to 1:1000; 4 h at 37 °C. Right panel: Time-dependent proteolysis of FN by KLK12, from 1 min to 5 h at 37 °C (E:S molar ratio: 1:50). (C) In control experiments, KLK12 was incubated with either E64 (100 µM), PMSF (0.5 mM) or EDTA (0.5 mM) and with FN. (D) Dose-dependent proteolysis of cellular FN isoforms (cFN and FN-EDB) by KLK12. E:S molar ratio: from 1:5 to 1:50; 4 h at 37 °C. The band corresponding to recombinant human KLK12 is indicated by an arrowhead when visible, and bands corresponding to proteolytic products are indicated by arrows.