Figure 2.

Inhibition of bleomycin (BLM)-induced epithelial–mesenchymal transitions (EMT) in ATII cells by caveolin-1 scaffolding domain peptide (CSP). A: ATII cells isolated from WT mice were treated with PBS or 40 μg/mL BLM alone or BLM with 10 nmol/L CSP or control peptide (CP) for 72 hours in culture dishes at 37°C. The lysates were immunoblotted for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin antigens. The lysates from ATII cells treated with 2 ng/mL transforming growth factor β (TGF-β) were used as a positive control for comparison. The densities of individual bands were normalized against the corresponding densities of β-actin. The fold changes in proteins are presented as a bar graph. Differences between PBS control and BLM or BLM and BLM+CSP groups are statistically significant. B: ATII cells isolated from WT mice were exposed to PBS, Ad-EV, or Ad-TGF-β with or without CSP or CP in culture dishes. After 72 hours, the lysates were analyzed for changes in EMT markers by Western blot analysis. C: ATII cells isolated from WT mice were cultured in Matrigel-coated plastic dishes for 0 to 3 days at 37°C. The lysates were later analyzed for changes in SP-A, SP-B, and SP-C expression by Western blot analysis. D: WT mice were exposed to saline or 2 U/kg body weight BLM through intranasal instillation. After 24 hours, mice exposed to BLM were i.p. injected with or without 18.75 mg/kg body weight of CSP or CP. Three days after BLM injury, ATII cells were isolated and the lysates were immunoblotted for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin. The densities of individual bands were measured and normalized against the corresponding densities of β-actin. The fold changes in proteins are presented as a bar graph. E: WT mice were exposed to ambient air or PCS from 40 cigarettes (approximately 90 mg/m³ total solid particulates) using a mechanical smoking chamber over a 2-hour period for 5 days per week. After 4 weeks of PCS exposure, mice exposed to PCS were i.p. injected with or without 18.75 mg/kg body weight of CSP or CP once every week for 4 more weeks. After 20 weeks of PCS exposure, ATII cells were isolated, and the lysates were tested for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1 and β-actin by Western blot analysis. F: Total RNA isolated from ATII cells of mice treated with saline, BLM, BLM+CSP, or CP were analyzed for changes in the expression of E-cadherin, collagen-I, ZO-1, and α-SMA mRNAs by quantitative real-time PCR. Changes in their expression levels were normalized to the corresponding levels of β-actin transcripts. The data were presented relative to that of saline-treated control groups. G: WT mice were exposed to saline, Ad-EV, or Ad-TGF-β by intranasal instillation. Twenty-four hours later, mice transduced with Ad-TGF-β were treated with or without CSP or CP. ATII cells were isolated from these mice 72 hours after exposure to TGF-β and analyzed for changes in EMT markers by Western blot analysis. H: WT mice treated with Ad-EV or Ad-TGF-β alone or Ad-TGF-β with CSP or CP as described in G were euthanized 21 days after initial transduction with Ad-TGF-β. The lung homogenates were analyzed for total hydroxylproline contents to assess changes in lung fibrosis. I: Mice were i.v. (via orbital plexus) injected with or without lentivirus expressing p53-binding or non–p53-binding control chimeric 3′-UTR sequences of uPA/uPAR/PAI-1 mRNA 3′-UTR as described elsewhere.¹⁴ Twenty-four hours later, the mice were exposed to BLM, and ATII cells were isolated 72 hours after inception of BLM injury. ATII cells isolated from mice exposed to saline were used as control for comparison. The lysates were tested for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin. ^∗∗∗∗P < 0.0001 versus control.