Fig 1: Smad3 regulates a transcriptional program associated with vascular outward expansion in conjunction with Sox9 and HoxB2.(a) Significantly enriched GO biological processes represented by Smad3 differentially regulated genes. (b) Enrichment of human disease terms associated with differentially regulated genes. (c) Relative expression of LOX and MFAP5 in HCASMC in the presence and absence of TGFβ, in cells transfected with siRNA against SMAD3 (SMAD3-KD) or non-targeting control, two tailed t-test. (d) HOMER analysis results for motifs enriched in promoter regions of genes differentially regulated in Smad3ΔSMC mice. (e) Expression of MMP3 and MFAP5 in control and SOX9 or HOXB2 knockdown HCASMC, two tailed t-test. (f) Co-immunoprecipitation of His-tagged HOXB2 (lane 4/5) or Flag-tagged SOX9 (lane 6) with endogenous SMAD3 compared with IgG or control vector (lanes 2/3). (g) Proximity ligation assay demonstrates close proximity of HOXB2-SMAD3 and SOX9-SMAD3 in the nucleus of HCASMC. (h) Relative luciferase activity of MFAP5-Luc in HEK-293 cells transfected with HOXB2 and SOX9 expression vectors in the absence (left) or presence (right) of SMAD3 siRNA (SMAD3-KD) or control siRNA (control), two-tailed t-test. All error bars represent 95% CI of mean. HEK-293 authenticity was validated upon receipt from the ATCC, and assessed periodically during the course of this study by cell morphometry and PCR evaluation of lineage markers. Abd, abdominal.
Fig 2: A schematic model based on our results showing the role of E2 in DCIS metastasis to IDC. E2 induced basement membrane disruption in breast glandular ducts by promoting MMP-3 and IL-1ß secretion via the GPER signaling pathway. In turn, the secreted IL-1ß activated the IL-1R1/MyD88 signaling pathway to increase IL-1ß and MMP-3 expression. Finally, in our model, estradiol induces apoptosis and pyroptosis of epithelial cells, thereby disrupting the glandular ducts and promoting DCIS metastasis to IDC.
Fig 3: Effect of E2-induced MMP-3 secretion on cell adhesion and basement membrane. (a) Western blotting of MCF-10A cells showing pro- MMP-3 following treatment with 32 nM E2 for 0, 24, and 48 h. The presented blots were cropped. Full-length blots are presented in Supplementary Fig. 7. (b) MMP-3 activity assay of MCF-10A cells for measuring MMP-3 activity in cell culture media following treatment with 32 nM E2 for 0, 24, and 48 h. Four independent experiments were performed. Bars represent +/-SD. (c) MMP-3 activity assay of MCF-10A cells following treatment with 32 nM E2 with or without 1.6 µM N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), which is a MMP-3 inhibitor. Four independent experiments were performed. Bars represent +/-SD. (d) Representative images of MCF-10A cells treated with 32 nM E2 or 32 nM E2 and NNGH at 200, 800, or 1600 nM for 3 days to detect cell junctions by immunofluorescence using the pan-cadherin antibody (green). Blue staining, Hoechst. Scale bars = 5 µm. (e) Confocal images of MCF-10A cells in a 3D culture treated with 32 nM E2 (first row) or 32 nM E2 and 1600 nM NNGH (second row) for 14 days to investigate the basement membrane via staining with laminin V antibody (red) and cell junctions via the pan-cadherin antibody (green). Reconstruction structures of the acini are shown in the right panel by Hoechst (blue) and laminin V (red). Arrows indicate the collapsed portion of the basement membrane. Scale bars = 5 µm.
Fig 4: Transmission electron microscopy (TEM) analysis of ECM remodelling in outflow tissues. Semi-thin sections of the iridocorneal angle in mouse eyes treated with either (A) AAV-Null or (B) AAV-MMP-3. AAV-MMP-3 treated eyes show greater inter-trabecular spaces in outer trabecular meshwork (TM) than controls. Scale bar denotes 50 μm. (C,D) Transmission electron micrograph of the inner wall of Schlemm’s Canal (SC) and the outer TM. (C) Control eye illustrating normal attachment between foot-like extensions of the inner wall endothelium and subendothelial cells (arrowheads), as well as with the discontinuous basement-membrane material underlying the inner wall endothelium (arrows). (D) Representative TEM image of an MMP-3 treated eye showing a disconnection of the inner wall endothelium from the subendothelial cells and the ECM (arrowheads). The widened subendothelial region lacks basement-membrane material and other ECM components. (E,F) Higher magnification of the inner wall of a treated eye. (E) Foot-like extensions of the inner wall endothelium (E) have disconnected from the subendothelial cells and the ECM (arrowheads), and the lack of ECM in this region is shown. (F) In other regions of treated eyes, clumps of presumably degraded ECM-material are localised underneath the inner wall of SC (asterisk). Such clumps of ECM are not present in controls. Scale bars are denoted on each image. CB = ciliary body, I = iris, R = retina. (G). Morphometric measurements of the optically empty space immediately underlying SC from four regions of contralateral eyes treated with AAV-MMP-3 (red data points) or AAV-Null (blue data points). Bars indicate average values for each eye. Contralateral eyes are presented immediately next to one another.
Fig 5: Effect of recombinant human MMP-3 on paracellular permeability in HTM and SCEC cell monolayers. SCEC and HTM cells were treated with 10 ng/ml recombinant MMP-3 for 24 h, using PBS and inactivated MMP-3 (incubation with TIMP-1, MMP(−)) as vehicle and negative controls respectively. (A) SCEC and (B) HTM both show reductions in TEER values after treatment of 4.6 [2.9, 6.2] and 5 [2.2, 7.8] Ohms.cm2 respectively. Permeability to a 70 kDa dextran was increased in treated cells (MMP (+)) in both (C) SCEC and (D) HTM. (E) An average viability of 85% was expected for SCEC with MMP-3 concentrations up to 36 ng/ml. (F) 85% viability is retained on average in HTM cells at concentrations up to 151 ng/ml MMP-3. A, C and E in blue represent SCEC data, whereas B, D and F in red represent HTM data.
Supplier Page from Abcam for MMP-3 Activity Assay Kit (Fluorometric)