FIGURE 7.

Inhibition of β-catenin/TCF-dependent transcription by ICAT antagonizes expression of markers of AT1 cell differentiation. AT2 cells were isolated from rat lungs and infected (in duplicate wells) with adenoviral constructs on Day₀ and lysed on Day₃ of culture. A, total β-catenin, T1α (AT1 marker), pan-cytokeratin (epithelial marker), vimentin (fibroblast marker), β-tubulin (loading control), and HA and Myc tags for Ad-S37A-β-catenin-HA and Ad-myc-ICAT-GFP, respectively, were detected by Western blot as described under “Materials and Methods.” Duplicate infections are shown in parallel lanes. B, Western blot for RAGE (AT1 marker) and GAPDH (loading control) from adenovirus-infected cells (Day₃). Note that ICAT decreases the expression of T1α and RAGE; constitutively active β-catenin does not apparently alter AT2 transdifferentiation, despite the fact that the S37A-β-catenin adenovirus is competent to up-regulate the β-catenin/TCF target gene, AXIN2 (not shown). This experiment was carried out using nine different rat AT2 isolates on different days; six of nine experiments showed an inhibition of >2-fold. C, graph represents normalized densitometry values of scans from T1α immunoblots. Densitometry was performed using the CanoScan software (Canon) and analyzed using ImageJ (National Institutes of Health). Error bars reflect ± S.E. The two-tailed p value is 0.0519 (*). D, live cell GFP-fluorescence images of control, Ad-GFP-infected, and Ad-ICAT-GFP-infected AT2 cells 72 h post-infection. Note that many cells expressing Ad-ICAT-GFP are viable as assessed by GFP fluorescence and the exclusion of Trypan blue (not shown).