JCS03232 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Confocal images obtained from cryostat sections of first-trimester placenta using triple-labeling staining. Nuclei are visualized with Sytox Green and pseudocoloured in blue, EMILIN1 is detected with Alexa Fluor® 633 and pseudocoloured in green, trophoblast cells are stained with anti-cytokeratin 7 (Alexa Fluor® 546 anti-mouse). Images were acquired with a Leica TCS SP2 confocal system (Leica Microsystems), using the Leica Confocal Software (LCS) and a 40× fluorescence objective on a Leica DM IRE2 microscope. In A the image and its magnification to the right are shown as the maximal projection of the summation of total z-sections. Arrows indicate trophoblasts. Images in B are 0.4 μm z-sections at different levels of the stack as indicated. Asterisks indicate three spiral arteries. Bar, 75 μm.

• Supplemental Figure 2 -

  Fig. S2. Morphology of cells adhering to ECM substrates. (A) HTR-8/SVneo cells attached onto EMILIN1 do not form stress fibers nor focal adhesions. Cells were plated for 30 minutes on substrate-coated coverslips, fixed, and stained with Texas-red phalloidin (Molecular Probes) and anti-paxillin (upper panel) or anti-FAK (lower panel) antibodies, followed by FITC-conjugated rabbit anti-mouse IgG. Actin-containing stress fibers and focal-adhesion formation are evident only in cells attached to FN and VN. Cells attached to EMILIN1 show subcortical actin organization and diffuse positivity for paxillin and FAK. (B) Time-course morphological analysis of cells adhering on FN (upper panel) or on EMILIN1 (lower panel). Cells were allowed to adhere on ECM substrates for different times and then treated to visualize paxillin (green) and actin (red). Note that cells plated on EMILIN1 did not form focal plaques even after 3 hours of adhesion, whereas on FN the focal contacts are visible in 20 minutes. Bar, 25 μm.

• Supplemental Figure 3 -

  Fig. S3. (A) Immunoblotting analysis of cellular extracts from several cell types: FN, fibronectin; EMI, EMILIN1; lane 1, HTR-8/SVneo; lane 2, DSC; lane 3, UtSMC; lane 4, HUVEC. (B) Confocal images obtained from cryostat sections of first-trimester placenta using triple-labeling staining. Nuclei are visualized with Sytox Green and pseudocoloured in blue, EMILIN1 is detected with Alexa Fluor® 633 and pseudocoloured in green, trophoblast cells are stained with anti-cytokeratin 7 (Alexa Fluor® 546 anti-mouse, red, upper panel, left), endothelial cells with anti-CD31 (red, upper panel, right) and stromal cells with anti-vimentin (red, lower panel). Images were acquired with a Leica TCS SP2 confocal system (Leica Microsystems), using the Leica Confocal Software (LCS) and a 40× fluorescence objective on a Leica DM IRE2 microscope. Bar, 75 μm.

• Supplemental Figure 4 -

  Fig. S4. MMP production profiles of HTR-8/SVneo and DSC cells. (A) Zymography of conditioned media obtained from DSC and HTR-8/SVneo cells for detection of MMP-2 and MMP-9 (upper panel). Western blotting analyses of supernatants for detection of MMP-1, MMP-3, MMP-11 and of cell lysates for MT1-MMP. Untreated or cells treated for 24 hours with TPA or TNF-α to evaluate the effect of 24-hour stimulation on the production of MMPs were used. (B) Inhibition of haptotaxis. GM6001 (10 μM) or α4 function-blocking mAb P1H4 were added alone or together to block migration towards gC1q1, evaluated after 1 hour and 4 hours from the beginning of migration. P values were determined versus control samples.