(A) Western blot analysis of phospho-Akt and S6K1 in the absence or presence of the PI3K inhibitor wortmannin (WT) in response to control fibroblast attachment to monomeric collagen in the absence of serum

One representative example is shown. (B) Western blot analysis of phospho-Akt in control fibroblasts preincubated with the indicated integrin blocking antibody and plated on monomeric collagen. (C) Fibroblasts were plated on tissue culture plates coated with monomeric collagen and blocked by BSA. Cells were allowed to spread for 30 min. Cell areas of a random 150 cells were quantified by tracing the cell border using ImageJ software (available at ). (D) Western blot analysis of phospho-Akt and S6K1 in response to control fibroblast attachment to polymerized collagen in the absence of serum. (E) Western blot analysis of phospho-Akt and S6K1 in control fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media. One representative example is shown. (F) GD25, GD25 β1, and GD25 α2β1 fibroblasts were cultured on monomeric (left) or polymerized (right) collagen in the presence of serum, and cell numbers were quantified. (G) Proliferation assay of control fibroblasts expressing dominant-negative Akt (Ad-DN-Akt; left) or constitutively active p110 subunit of PI3K (Ad-PI3Kp110; right) and cultured on monomeric or polymerized collagen in growth factor–replete media, respectively. (top left) Western blot analyses of phospho- and total Akt. (bottom left) Shown is the percent change in cell growth. *, P < 0.01 and 0.001 for the percent change in fibroblast growth on monomeric collagen that was significantly less in control fibroblasts expressing dominant-negative Akt compared with cells expressing control vector at days 3 and 6, respectively. (top right) Western blot analyses of phospho- and total Akt. (bottom right) Shown is the percent change in cell growth. *, P < 0.01 and 0.0001 for the percent change in fibroblast growth on polymerized collagen that was significantly greater in control cells expressing constitutively active p110 subunit of PI3K compared with control vector at days 3 and 6, respectively. Error bars represent SEM. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A) Western blot analysis of total PTEN and phospho-Akt and S6K1 in IPF fibroblasts expressing wild-type PTEN (Ad-wtPTEN) or empty vector (Ad-GFP) and cultured on polymerized collagen in growth factor–replete media

(B) eIF4F activity (RLUC luminescence) in IPF fibroblasts expressing wild-type PTEN or control vector and cultured for 3 d on polymerized collagen. *, P < 0.0001 for significantly less eIF4F activity in IPF fibroblasts expressing wild-type PTEN compared with control. (C) Quantification of DNA synthesis by BrdU incorporation in IPF fibroblasts expressing wild-type PTEN or empty vector and cultured on polymerized collagen in growth factor–replete media for 3 d. Shown is the percentage of BrdU-positive IPF fibroblasts. *, P < 0.0001 for the percentage of BrdU-positive IPF fibroblasts expressing wild-type PTEN that was significantly less compared with control. Error bars represent SEM. Data are representative of three independent experiments from one IPF cell line.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A and B) Western blot analysis of total PTEN in control (A) or IPF (B) fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media

One representative sample out of five control and five IPF cell lines examined is shown. (C and D) PTEN phosphatase assay showing the percent change in PTEN activity in control ( = 3; C, left) or IFP ( = 3; D, left) fibroblasts cultured on polymerized or monomeric collagen in growth factor–replete media compared with cells cultured under proliferation-prohibitive conditions. *, P < 0.001 for PTEN activity in control fibroblasts on polymerized collagen that was significantly higher at day 3 compared with monomeric collagen; P < 0.01 for PTEN activity in IPF fibroblasts on polymerized collagen that was significantly less on day 3 compared with monomeric collagen. Shown is PI3K activity in control (C, right) and IPF (D, right) fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media for 3 d. *, P < 0.005 for PI3K activity in control fibroblasts cultured on polymerized collagen that was significantly less at day 3 compared with monomeric collagen; or P < 0.05 for PI3K activity in IPF fibroblasts cultured on polymerized collagen that was significantly higher at day 3 compared with monomeric collagen. Error bars represent SEM. (E) PTEN protein levels in membrane and cytosolic fractions of cell lysates were measured by Western blot analysis. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

Control fibroblasts were transfected with PTEN or control siRNA and cultured on polymerized collagen in serum

(A) Western blot analysis of total PTEN levels. (B) Western blot analysis showing PTEN protein expression in response to increasing concentrations of PTEN siRNA. (C) Proliferation assay of control fibroblasts on polymerized collagen in serum-replete media. After 6 d, there was a significant increase in proliferation on polymerized collagen in cells treated with PTEN siRNA compared with control siRNA (*, P < 0.04). Error bars represent SEM. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A) Western blot analyses of phospho-Akt and S6K1 in response to IPF fibroblast attachment to monomeric collagen in the absence of serum

One representative example is shown. (B) Western blot analysis of phospho- and total Akt in response to treatment of control and IPF fibroblasts with TS2/16 β1 integrin activating antibody. (C) Control ( = 3) or IPF ( = 3) fibroblasts were stained with anti–β1 integrin antibodies and analyzed by FACS. Anti–β1 integrin antibodies used were against ligand-induced binding sites (9EG7), activating sites (TS2/16), or total β1 integrin regardless of activation state (P5D2). (D) Control and IPF fibroblasts were plated on monomeric collagen. Shown is a Western blot analysis of phosphorylated FAK (tyr397). Data are representative of three independent experiments from one control and one IPF cell line. (E) PI3K activity in control and IPF fibroblasts plated on tissue culture dishes and treated with TS2/16 β1 integrin activating antibody. Data are representative of three independent experiments from one IPF cell line. (F) Western blot analysis of phospho-Akt and S6K1 in response to IPF fibroblast attachment to polymerized collagen. One representative example is shown. (G) Western blot analysis of phospho-Akt in IPF fibroblasts preincubated with the indicated integrin blocking antibody and plated on polymerized collagen. Data are representative of three independent experiments from one representative IPF cell line. (H) Western blot analysis of phospho-Akt and S6K1 in IPF fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media. Data are representative of three independent experiments from five IFP cell lines. (I) Proliferation assay of IPF fibroblasts expressing dominant-negative Akt (left) or treated with 100 nM rapamycin (right) and cultured on polymerized collagen in growth factor–replete media. Shown is the percent change in cell growth. *, P < 0.01 and 0.0001 for the percent change in cell growth on polymerized collagen that was significantly less in IPF fibroblasts expressing dominant-negative Akt at days 3 and 6, respectively; or P < 0.002 or 0.001 for the percent change in cell growth on polymerized collagen that was significantly less in IPF fibroblasts treated with rapamycin at days 3 or 6, respectively. Error bars represent SEM. Data are representative of three independent experiments from one IPF cell line. (J) IPF fibroblasts were preincubated with the indicated integrin blocking antibody and plated on polymerized collagen in growth factor–replete media in the presence of antibody. BrdU staining as a measure of DNA synthesis was assessed at 24 h. Error bars represent SEM. Data are representative of three independent experiments from three IPF cell lines.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p