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. 2015 Jan;185(1):55–68. doi: 10.1016/j.ajpath.2014.08.027

Figure 5.

Figure 5

The contribution of plasminogen activator inhibitor (PAI-1) in bleomycin (BLM)-induced ATII cell epithelial–mesenchymal transitions (EMT). A: ATII cells isolated from PAI-1–deficient mice were treated with PBS, BLM, BLM+CSP, or BLM+CP in vitro. The lysates were immunoblotted for changes in collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin expression. The lysates from ATII cells lacking PAI-1 expression that were treated with transforming growth factor β (TGF-β) were used as a control. The individual densities of bands were quantitated and normalized to the corresponding values of β-actin. The fold changes in protein expression are presented as a bar graph. Differences between treatments versus control are not statistically significant. B: Mice lacking PAI-1 expression were treated with saline or BLM, and 24 hours later, mice exposed to BLM were i.p. injected with or without caveolin-1 scaffolding domain peptide (CSP) or control peptide (CP). Three days after initiation of BLM injury, ATII cells were isolated from saline, BLM-, BLM+CSP-, or BLM+CP-treated mice. The lysates were examined for the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin by Western blot analysis. The densities of bands were normalized against the corresponding levels of β-actin. The bar graph represents the fold change of proteins. C: Total RNA isolated from ATII cells of PAI-1–deficient mice exposed to saline, BLM, BLM+CSP, or CP were analyzed for changes in the expression of E-cadherin, ZO-1, collagen-I, and α-SMA mRNA levels by real-time PCR. The data were normalized with corresponding levels of β-actin mRNA. The fold changes of RNA are presented as a bar graph. D: ATII cells isolated from PAI-1–deficient or uPA-deficient mice were treated with lentivirus expressing uPA or PAI-1 shRNA. ATII cells obtained from both uPA-deficient and PAI-1–deficient mice exposed to nonspecific shRNA or naive ATII cells were used as controls for comparison. Because BLM injury causes EMT in uPA-deficient mice, whereas those lacking PAI-1 expression resist BLM-induced EMT, ATII cells from uPA-deficient mice treated with or without PAI-1 shRNA or control shRNA were later exposed to BLM for 72 hours. The lysates from PAI-1–deficient and uPA-deficient ATII cells were analyzed for changes in EMT markers by Western blot analysis.