Kim et al. 10.1073/pnas.0605669103. |
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Supporting Text
Fig. 7. Loss of alveolar epithelial cell (AEC) apical-basal polarity in idiopathic pulmonary fibrosis (IPF) lung. IPF lung biopsy stained with pro-surfactant protein-C (pro-SPC) and integrin a3. AECs typically have a basilar distribution a3 staining. Several pro-SPC-positive AECs are found within the interstitium and have lost apical-basal polarity (arrows).
Fig. 8. Loss of pro-surfactant protein-C (pro-SPC) and E-cad staining on Fn. alveolar epithelial cells (AECs) from triple transgenic mice were isolated and cultured on Mg/Col- (A, C, and E) or Fn-coated (B, D, and F) slides for 1 week, then stained with X-gal (A and B) followed by immunostaining for pro-SPC (red, C and D) and E-cad (green, E and F). X-gal-positive cells maintain pro-SPC and E-cad on Mg/Col but lose these epithelial markers on Fn.
Fig. 9. Lung responses to active TGF-b1 overexpression. (A) Lung section from a triple transgenic mouse 3 weeks after AdTGF-b1 injury stained with X-gal, then picosirius red demonstrating b-gal-positive cells within a fibrotic region of lung. (B) Immunoblot of whole lung lysate 3 weeks after AdTGF-b1, lysate from freshly isolated AECs and murine fibroblasts are used as controls. (C) Ten days after AdTGF-b1 injury whole lung single cell preparation from triple transgenic were maintained on Mg/Col for one week, then stained with X-gal. Clusters of X-gal-positive epithelial cells are shown (arrowheads) as well as X-gal-positive cells exhibiting a mesenchymal phenotype (arrow). (D) High magnification (´60) view of X-gal-positive cells from injured lung.
Fig. 10. Vimentin specificity and staining on Fn. Primary murine alveolar epithelial cells (AECs) (A) or murine fibroblasts (B) were immunostained for vimentin, demonstating specificity of staining limited to fibroblasts. AECs from triple transgenic mice were maintained on Fn for 1 week, then resuspended and stained with X-gal (C) followed by immunostaining for vimentin (D), demonstrating vimentin staining in X-gal-positive cells.
Fig. 11. Distribution of Smad2/3 in wild-type and b6-null alveolar epithelial cells (AECs) on Fn. (A and B) Wild-type AECs cultured on Fn for 2 days then stained for Smad2/3 (A) and overlaid with dapi staining (B) demonstrating nuclear localization. (C and D) b6-null AECs cultured on Fn for 2 days stained for Smad2/3 (C) and overlaid DAPI staining (D) demonstrating diffuse Smad2/3 staining. (E) Percentage of wild-type (±SB431542, 10 mM) and b6-null AECs with a nuclear distribution of Smad2/3 after 2 days in culture on Fn-coated slides.
Supporting Text
Materials
. Mouse anti-vimentin, a-smooth muscle actin, b-tubulin and b-actin were purchased from Sigma (St. Louis, MO). Rabbit anti-human prosurfactant protein C and N-cadherin were obtained from Chemicon (Temecula, CA). Rat anti-E-cadherin was purchased from Zymed. Mouse anti-b-galactosidase was purchased from Promega (Madison, WI). Rabbit anti-phospho-Smad2 was purchased from EMD Biosciences (La Jolla, CA). Rabbit anti-Smad3/2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Murine anti-TGF-b1 (1D11) was obtained from Dean Sheppard (University of California, San Francisco, CA). Murine TGF-bRII-Fc was obtained from Paul Weinreb (Biogen Idec, Cambridge, MA). Sheep anti-mouse PAI-1 was purchased from American Diagnostica (Stamford, CT). Biotin conjugated anti-CD45 and anti-CD16/32 were purchased from BD Biosciences. Dynabeads M-280 streptavidin magnetic beads were purchased from Dynal Biotech (Oslo, Norway). Purified human TGF-b1 was purchased from R & D Systems (Minneapolis, MN). Purified human KGF was from PeproTech (Rocky Hill, NJ). The selective TGF-b1 receptor kinase inhibitor SB431542 was purchased from Sigma. Matrigel was purchased from BD Biosciences, type I rat tail collagen was purchased from Sigma, and purified human fibronectin was purchased from Chemicon.Type II Cell Isolation and Culture.
Murine alveolar epithelial type II cells were isolated from adult mice (6-12 weeks old) following the method of Corti et al. (1) with minor modifications. Cell viability was assessed by trypan blue exclusion and purity of alveolar type II cells was assessed by immunofluorescence staining for pro-SPC. Typically, 5-10 ´ 105 cells containing 90-95% type II pneumocytes were isolated from a single mouse consistent with previous reports. Cells were cultured on tissue culture plates and chamber slides coated with Mg, supplemented with 5% collagen type I and 25% SABM (Cambrex, East Rutherford, NJ), or coated with Fn, 100 mg/ml in PBS. Cells were maintained in SAGM without hydrocortisone containing 5% charcoal/dextran-treated FBS (HyClone) and 10 ng/ml KGF in a 37°C, 5% CO2 incubator as described (2). The media was replaced after 1 day, then cultured for an additional 1-6 days. In some cases, the cells were cultured in the presence of the selective TGF-b1 receptor kinase inhibitor, SB431542 (10 mM), the TGF-b1 blocking antibody 1D11 (100 mg/ml), or TGF-bRII-Fc (5 mg/ml) versus appropriate control (vehicle or control IgG). After 2-7 days, cells were analyzed as indicated.Whole-Lung Single-Cell Suspensions
. Single cell suspension was obtained from murine lungs as described (3) with minor modifications. Briefly, lungs were inflated with dispase, allowed to collapse, and then placed in 1 ml of dispase while gently agitating at room temperature for 45 min. Lungs were minced to ~1-mm pieces and resuspended in 2 ml of dispase containing collagenase (2 mg/ml) and DNase (50 mg/ml). Digested lungs were resuspended in DMEM 10% FBS and sequentially filtered through 70- and 40-mm filters. Cells then underwent negative selection using anti-CD45 and anti-CD16/32 antibodies. Cells were either cultured or immediately fixed and stained with X-gal in suspension. Cells stained with X-gal in suspension were then centrifuged onto superfrost-coated slides (Fisher) and immunostained.