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1.
Figure 4

Figure 4. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP is retained on collagen and ACLP−/− lung fibroblasts have normal adhesion to collagen. A: ACLP Western blot of total proteins extracted from wild-type lung fibroblasts cultured for 18 hours on either collagen gels (Col gel) or tissue culture plastic (plastic). B: Lung fibroblasts were cultured for 18 hours on either collagen gels (Col gel) or tissue culture plastic (plastic) followed by extraction of cell and ECM protein fractions as indicated in the Materials and Methods section. ACLP Western blot of cell and ECM fractions: lanes 1 and 2 cell and ECM proteins, respectively, from fibroblasts cultured on collagen; lanes 3 and 4 cell and ECM proteins, respectively, from fibroblasts cultured on tissue culture plastic. C: Adhesion of ACLP+/+ and ACLP−/− lung fibroblasts was measured as described in Materials and Methods. ACLP+/+ on Col I (closed square), ACLP−/− on Col I (open square), ACLP+/+ on BSA (closed circle), ACLP−/− on BSA (open circle).

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
2.
Figure 1

Figure 1. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP levels increase in mouse lungs after bleomycin injury. A: Wild-type mice (n = 5 each group) were treated with either 50 μl of intratracheal normal saline or 0.0011 U/g body weight of bleomycin. Lungs were harvested after 7 or 28 days, fixed, and immunostained for ACLP (brown, black arrows), stained with Masson’s trichrome for collagen (Trichrome, blue, white arrow), or immunostained for α-SMA (red, gray arrows). Data are representative of parallel 5 μm sections for each condition. Arrows indicate areas of specific immunostaining (magnification = original ×200). B: ACLP and β-actin Western blots of whole-lung protein extracts from wild-type mice 28 days after treatment with saline or bleomycin (Bleo). C: Densitometric quantitiation of ACLP Western blots of whole-lung protein extracts from wild-type mice 28 days after treatment with saline or bleomycin (n = 4 each group).

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
3.
Figure 3

Figure 3. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP+/+ and ACLP−/− mice have similar early inflammatory responses to bleomycin injury. A: Wild-type (+/+) and ACLP-deficient (−/−) mice were treated with either saline or bleomycin (Bleo) (n = 3 each group). After 7 days BALs were performed on anesthetized mice using 1.5 ml of PBS. After staining, BAL macrophages (black bars), neutrophils (gray bars), and lymphocytes (open bars) were counted and the cell counts expressed per ml of recovered BAL fluid. B: Wild-type (ACLP+/+) and ACLP-deficient (ACLP−/−) mice were treated with bleomycin (n = 4 each group). After 7 days, harvested lungs were immunostained for CD45 (brown) and the staining quantified by measuring the area of brown staining per ×100 microscope field; the data are expressed as % staining per ×100 field (C). Wild-type (+/+) and ACLP-deficient (−/−) mice were treated with either saline or bleomycin (Bleo) (n = 3 each group). After 7 days, BALs were performed on anesthetized mice using 1.5 ml of DMEM. Active TGF-β1 in the BAL fluid was assayed and expressed as ng/ml of BAL fluid (D) (#N.D. = none detected). All data are mean values ± SE.

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
4.
Figure 2

Figure 2. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP−/− mice accumulate fewer α-SMA-positive fibroblasts and less collagen in the lung after bleomycin injury compared with wild-type mice. A: Wild-type (ACLP+/+) and ACLP-deficient (ACLP−/−) mice were treated with intratracheal bleomycin (n = 6 each group). After 28 days harvested lungs were immunostained for α-SMA (red indicated by arrows) or stained with Masson’s trichrome (Trichrome, blue) to detect collagen. Data are representative of 5 μm sections for each condition. B: Wild-type (+/+, filled bar) and ACLP-deficient (−/−, open bar) mice were treated with bleomycin (Bleo) (n = 6 each group). After 28 days, harvested lungs were immunostained for α-SMA and the staining quantified by measuring the area of red staining per ×100 field; the data are expressed as % staining per ×100 field. C: Wild-type (+/+, filled bars) and ACLP-deficient (−/−, open bars) mice were treated with either saline or bleomycin (Bleo) (n = 6 each group). After 28 days harvested lungs were assayed for whole-lung hydroxyproline content and expressed as hydroxyproline (μg/mg lung weight). Data are mean values ± SE (#P < 0.05 for bleomycin-treated compared with saline-treated ACLP+/+ mice and *P = NS for bleomycin-treated compared with saline-treated ACLP−/− mice).

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
5.
Figure 7

Figure 7. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP−/− lung fibroblasts have a defect in cell proliferation on collagen matrix. Wild-type (closed circle) and ACLP-deficient (open circle) primary lung fibroblasts were cultured under serum-free conditions on restrained collagen gels (A) or tissue culture plastic (B) in 24-well dishes at a starting density of 1 × 105 cells/well. At the indicated time points, cells were extracted from collagen gels by collagenase digestion (A) or removed from plastic wells using trypsin (B). Cells were then counted using a Coulter particle counter and expressed as cells extracted/gel (A) or cells removed/well (B). All data are mean values ± SE. All experiments were performed at least three times with different primary cell cultures.

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
6.
Figure 8

Figure 8. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP is expressed by human fibroblasts and levels are increased in human fibrotic lung tissue. A: IMR-90 cells were cultured in the absence (−) or presence (+) of 10 ng/ml TGF-β. After culture for the indicated amount of time, cell proteins were extracted, subjected to SDS-PAGE and Western blots were performed to detect ACLP, α-SMA, and tubulin. B: Surgical specimens from normal human lung (n = 6) and from patients with idiopathic pulmonary fibrosis [IPF] (n = 6) were immunostained with either control (pre-immune) rabbit serum or anti-ACLP rabbit serum (brown) or stained with Masson’s trichrome (Trichrome, blue) to detect collagen. Data are representative serial 5-μm sections for each condition. Black arrows indicate regions of ACLP expression; white arrows indicate collagen rich areas. V: blood vessel.

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
7.
Figure 6

Figure 6. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

The discoidin-like domain of ACLP promotes fibroblast spreading on collagen and increases collagen matrix contraction by fibroblasts. A: Purified His-tagged DLD protein was analyzed by SDS-PAGE and stained as described in the Materials and Methods. Lane 1, MW marker. Lane 2, 1 μg aliquot of purified DLD. B: Phase contrast photomicrographs of ACLP-deficient primary lung fibroblasts cultured under serum-free conditions on restrained collagen gels at a density of 1 × 105 cells/gel for 24 hours in the presence of 10 μg/ml BSA (Control) or 10 μg/ml of the discoidin-like domain (DLD) of ACLP (+DLD). The arrows indicate cells with enhanced spreading and membrane extensions; viewed at ×100. C: ACLP-deficient (ACLP−/−) and (D) wild-type (ACLP+/+) primary lung fibroblasts were cultured under serum-free conditions on restrained collagen gels at a density of 2 × 105 cells/gel for 24 hours in the presence of 0 (closed circle), 1 (open circle), 10 (closed box), or 50 (open box) μg/ml DLD. The collagen gels were then released and allowed to contract for the indicated amount of time. Collagen gel contraction is expressed as percent original gel size. All data are mean values ± SE. Data are from one of three experiments all showing similar results.

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.
8.
Figure 5

Figure 5. From: Aortic Carboxypeptidase-Like Protein Is Expressed in Fibrotic Human Lung and its Absence Protects against Bleomycin-Induced Lung Fibrosis.

ACLP−/− lung fibroblasts have a defect in cell spreading and contraction of a collagen matrix. A: Phase contrast photomicrographs of wild-type (ACLP+/+) and ACLP-deficient (ACLP−/−) primary lung fibroblasts cultured on restrained collagen gels for 24 hours at a density of 2 × 105 cells/gel. Clustering of ACLP−/− fibroblasts is marked by arrows; viewed at ×40. B: Wild-type (ACLP+/+) and ACLP-deficient (ACLP−/−) primary lung fibroblasts were cultured on restrained collagen gels for 24 hours at a density of 2 × 105 cells/gel. The gels were then released, allowed to contract for 6 hours and photographed; arrows mark gel edges. C: Time course of collagen gel contraction by wild-type (closed circle) and ACLP−/− (open circle) lung fibroblasts. Cells were cultured and collagen gels released and allowed to contract as in panel (B); gel contraction is expressed as a percentage of the original gel size. D: Primary lung fibroblasts were cultured and collagen gels released and allowed to contract as in panel (B). Twenty min before releasing gels, fibroblasts were cultured in the absence [wild-type (ACLP+/+, closed circle); ACLP-deficient (ACLP−/−, open circle)] or presence [wild-type (ACLP+/+ + LPA, closed inverted triangle); ACLP-deficient (ACLP−/− + LPA, open triangle)] of 10 μm lysophosphatidic acid. Gel contraction is expressed as a percentage of the original gel size. E: Wild-type (+/+) and ACLP-deficient (−/−) primary lung fibroblasts (2 × 105) were cultured on restrained collagen gels for 18 hours. Twenty minutes before releasing gels, cells were cultured in the absence or presence (+LPA) of 10 μm lysophosphatidic acid. After gel release and contraction for the indicated time points [time (min)], total cell proteins were immediately extracted and equal amounts subjected to SDS-PAGE followed by Western blotting using antibodies to phosphorylated myosin light chain (P-MLC) and total myosin light chain (MLC); data shown are from one of two experiments showing similar results.

Scott L. Schissel, et al. Am J Pathol. 2009 March;174(3):818-828.

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