Colocalization of TGF-β-RII and the fibroblast marker prolyl-4-hydroxylase

<p><b>Copyright information:</b></p><p>Taken from "Localization of TGF-β type II receptor and ED-A fibronectin in normal conjunctiva and failed filtering blebs"</p><p></p><p>Molecular Vision 2008;14():136-141.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2255025.</p><p></p> Native conjunctiva () and scarred filtering bleb specimens (, ) were double-stained for TGF-β-RII (green) and prolyl-4-hydroxylase (red). is a close-up of a portion of as indicated by the frame. Arrows point to a TGF-β-RII positive fibroblast, which are positive for both TGF-β-RII and prolyl-4-hydroxylase. Scale bar represents 50 µm (,) and 10 µm ()

Localization of α-smooth muscle actin (SMA) and the absence of lymphatic endothelium in scarred filtering bleb tissue

<p><b>Copyright information:</b></p><p>Taken from "Localization of TGF-β type II receptor and ED-A fibronectin in normal conjunctiva and failed filtering blebs"</p><p></p><p>Molecular Vision 2008;14():136-141.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2255025.</p><p></p> Serial sections were double labeled for TGF-β-RII (, , and green) and α-SMA (, ), O-linked sialoglycoprotein (, ), and PECAM-1 (, , red). α-SMA is colocalized to TGF-β-RII in stromal fibroblasts and perivascular cells (,,). Vascular structures expressing TGF-β-RII() were negative for markers of lymphatic endothelium ().Some collagen fiber autofluorescence is present in the red channel (, , ), Scale bar represents 50 µm

Localization of fibronectin in invadosomal degradation zones.

<p>HTM cells were pretreated with TGF-β for 3d, plated in the gelatine degradation assay and stained for fibronectin after 24 h. Confocal micrographs depict localization of gelatine and fibronectin. Close-up views of distinct areas (A, B, C) depict reticular fibronectin in a region of invadsomal gelatinolysis (arrow) and fibrillar strands of fibronectin which appear to originate at the edge of digestion zones (arrowheads). Scale bar represents 40 µm.</p

Effect of a ROCK inhibitor on TGF-β-induced invadosomes in HTM cells.

<p>(A) ECM degradation assay performed in the presence or absence of TGF-β2 or H1152 as indicated. (B) Close-up of the area marked in (A), depicting colocalization of cortactin and F-actin to sites of ECM degradation. (C) Quantitation of digested surface area in (A). Scale bar represents 40 µm (A), *** indicate p>0.001 in (C).</p

Extracellular matrix protein transcription.

<p>HTM cells were treated with vehicle or TGF-β2 in the presence or absence of H1152 for 3d. HPRT-normalized relative transcript levels for fibronectin (FN), collagen-1, -4 and -6 as detected by qPCR. Triplicate means ± SEM. Asterisks indicate significance of difference from controls ***p<0.001, ** p<0.01, * p<0.05; n.s. p≥0.05).</p

Effect of TGF-β2 on invadosomal proteolysis in HTM cells.

<p>The cells were treated with vehicle or TGF-β2 for 3d before plating in the ECM degradation assay. Confocal micrographs depict fluorescently labelled gelatine (green in A–D, grayscale in I–L) and F-actin (red in A–D, grayscale in E–H). TGF–β2 increased F-actin staining intensity (insert in F) and gain was reduced (in F–H) for image acquisition to avoid signal saturation. Scale bar represents 40 µm.</p

Primer pairs.

<p>Primer pairs.</p

Effect of TGF-β2 on MMP-2 activity in HTM cells.

<p>Cells were treated with vehicle or TGF-β2 for 3d, serum-starved for 15 h, conditioned starvation medium was collected, proteins were concentrated using spin-columns and subjected to zymography. MMP-2 renders two bands, a lower one for the cleaved active enzyme (arrowhead) and a higher one for the larger uncleaved protein, as indicated by the positive control (MMP-2 control).</p

Effect of a ROCK inhibitor on TGF-β-induced MMP-2 activity and protein expression.

<p>(A) Zymogram of conditioned media derived from HTM cells treated with vehicle or TGF-β in the presence or absence of H1152. First lane to the left depicts recombinant MMP-2 positive control. (B) Western Blot of conditioned media and cell lysate proteins.</p

Primer.

<p>Primer.</p