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1.
Fig. 5.

Fig. 5. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

Human lung fibroblasts utilize MMPs to degrade type I collagen. A: expression levels of the type I collagenolytic MMPs were assessed by semiquantitative RT-PCR. B: human lung fibroblasts were cultured atop a type I collagen substratum in the presence of 10% serum without protease inhibitors or in the presence of 100 μg/ml aprotinin, 100 μM E64, 5 μM BB-2516, or 20 μg/ml plasminogen. After 7 days, cells were lysed, and the substrata were stained with Coomassie Brilliant Blue. C: human lung fibroblasts were cultured atop Alexa Fluor-594-labeled type I collagen in the presence of the indicated inhibitors for 7 days, and subjacent collagenolysis was examined by confocal laser microscopy (scale = 20 μm).

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.
2.
Fig. 2.

Fig. 2. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

Matrix metalloproteinase (MMP)-mediated type I collagen degradation by pulmonary fibroblasts. A: fibroblasts were cultured in the center of type I collagen films in 10% serum under control (no inhibitor) conditions or in the presence of 100 μg aprotinin, 100 μM E64, or 5 μM BB-2516 for 7 days when cells were lysed and type I collagen substratum stained with Coomassie Brilliant Blue. B: hydroxyproline solubilization was quantified in the presence of the various protease inhibitors and presented as percent control measurements. Results presented as mean ± 1 SD (n = 3; *P = 0.001).

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.
3.
Fig. 1.

Fig. 1. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

Type I collagen degradation by mouse pulmonary fibroblasts. A: pulmonary fibroblasts were isolated from mouse lung, seeded atop type I collagen films, and viewed by phase contrast microscopy (left; scale = 50 μm) or immunostained with an antibody against α-smooth muscle actin (α-SMA) (right; scale = 30 μm). B: pulmonary fibroblasts were seeded atop type I collagen films in the absence or presence of 10% FCS or various stimuli. After 7 days, culture supernatant was collected and soluble hydroxyproline quantified. Cytokines were used at the following concentrations: platelet-derived growth factor-BB (PDGF, 10 ng/ml), transforming growth factor-α (TNF-α, 10 ng/ml), interleukin-1β (IL-1β, 5 ng/ml), or interferon-γ (IFN-γ, 10 ng/ml). Results are presented as the mean ± 1 SD (n = 4). C: collagen films were incubated alone or with pulmonary fibroblasts that were cultured in the center of a collagen gel for 3 days in the absence or presence of 10% FCS, lysed, with the collagen gel visualized by staining with Coomassie Brilliant Blue. D: cells were seeded atop Alexa Fluor-594-labeled type I collagen and counterstained either with calcein (left) or Alexa Fluor-488-labeled phalloidin and TOTO-3 iodide (right, scale = 20 μm).

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.
4.
Fig. 6.

Fig. 6. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

Human lung fibroblasts require MT1-MMP for type I collagenolysis. A: human lung fibroblasts were transfected with siRNAs directed against the transcripts encoding MMP1 and MMP14 or a scrambled MMP14 sequence (SCR), and levels of the various MMP transcripts were measured by semiquantitative RT-PCR. B: Western blot analysis demonstrating silencing of MT1-MMP protein expression in human pulmonary fibroblasts. C: human lung fibroblasts transfected with the indicated siRNAs were cultured atop Alexa Fluor 594-labeled type I collagen substrata for 7 days, when subjacent collagen degradation was examined by confocal laser microscopy (scale = 30 μm). D: human lung fibroblasts transfected with the indicated siRNAs were cultured atop type I collagen substrata for 7 days, and hydroxyproline levels in the culture supernatant were quantified (mean ± 1 SD; n = 3; *P = 0.0001 vs. SCR condition). E: human lung fibroblasts were transfected with the indicated siRNAs and cultured within a 3-D collagen matrix embedded within an outer, cell-free collagen gel in the presence of 10 ng/ml PDGF-BB for 10 days. White broken line indicates the interface of the cell-containing and cell-free gels, and the black broken line indicates the invasion front (scale = 200 μm).

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.
5.
Fig. 3.

Fig. 3. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

Membrane type-1 (MT1)-MMP is required for type I collagenolysis by pulmonary fibroblasts. A: RNA was isolated from wild-type (WT) mouse lung fibroblasts and mRNA levels of G3pdh-1, mouse collagenase A (McolA), Mmp2, Mmp8, Mmp13, Mmp14, and Mmp15 were measured by RT-PCR. B: pulmonary fibroblasts isolated from WT mice or mice with targeted genetic deficiencies in Mmp2, Mmp13, or Mmp14 were cultured in the center of type I collagen substrata for 7 days, when residual type I collagen was visualized by staining with Coomassie Brilliant Blue. C: mouse fibroblasts were cultured atop substrata of Alexa Fluor-594-labeled type I collagen for 7 days, and subjacent collagenolysis was examined by confocal laser microscopy (scale = 20 μm). Type I collagen degradation by each fibroblast strain was quantified by hydroxyproline measurement in the culture supernatant (mean ± 1 SD; n = 3; *P ≤ 0.0002). D: fibroblasts were cultured either atop a type I collagen substratum (left, scale = 50 μm) or embedded within a type I collagen hydrogel (right, scale = 200 μm) and cell morphology assessed after 7 days by phase contrast microscopy. E: hydroxyproline solubilization was measured WT fibroblasts under the indicated culture conditions as well at MT1-MMP-deficient fibroblasts cultured within 3-D type I collagen (mean ± 1 SD; n = 3; *P = 0.0015 compared with WT 3-D cells).

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.
6.
Fig. 4.

Fig. 4. From: Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers.

MT1-MMP is required for pulmonary fibroblast invasion of type I collagen extracellular matrix barriers. A: WT fibroblasts with or without 5 μM BB-2516 or MT1-MMP−/− fibroblasts were cultured atop type I collagen hydrogels in the upper chamber of a Transwell tissue culture plate insert with a chemotactic stimulus of 10 ng/ml PDGF-BB in the lower well for 3 days. Collagen gels were then fixed, sectioned, and stained with hematoxylin and eosin and examined by light microscopy (scale = 100 μm). Number of invading cells were quantified. A representative example of 3 individual experiments is presented. B: WT fibroblasts with or without 5 μM BB-2516 or MT1-MMP−/− fibroblasts were cultured within small (100 μl) collagen gels embedded in an outer, cell-free collagen gel, and invasion from the inner into the outer gel in the presence of 10% serum and 10 ng/ml PDGF-BB was monitored for 10 days. Invasion was assessed by phase-contrast microscopy (top, scale = 200 μm) or following staining with phalloidin (green, marking F-actin) or TOTO-3 iodide (red, marking nuclei, bottom, scale = 100 μm). Results are presented as the mean ± 1 SD (n = 3). C: WT fibroblasts with and without 5 μM BB-2516 and MT1-MMP−/− fibroblasts were cultured in the center of type I collagen substrata. Cell migration atop the 2-D substratum was monitored over 4 days by phase contrast microscopy (scale = 1 mm). Representative results of 3 independent experiments are presented. The migratory front is indicated by black lines.

R. Grant Rowe, et al. Am J Physiol Lung Cell Mol Physiol. 2011 November;301(5):L683-L692.

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