Tamoxifen-induced recombinase activity of Cre-ER^T2 in the lung tissues of SPC-Cre-ER^T2/TSC1^fx/fx transgenic mice.

<p>DNAs from the lung tissues of SPC-Cre-ER^T2/TSC1^fx/fx transgenic mice treated with vehicle or tamoxifen were examined by PCR to detect <i>TSC1</i> deletion as an indication of Cre-ER^T2 recombinase activity. M, DNA marker; +, a transgenic mouse with <i>TSC1</i> deletion; -, C57BL/6J mouse. Other lanes, offspring from F42 and F67 founders treated with vehicle (vehi) or tamoxifen (tam).</p

Generation of SPC-Cre-ER^T2 mice.

<p>A) Schematic map of SPC-Cre-ER^T2 expression cassette. HSPC-P, human surfactant protein C promoter; Cre-ER^T2, Cre coding sequence fused with a tamoxifen-inducible estrogen receptor. pA, a polyA sequence from SV40 virus. Cre-F primer binding sites, 561–579 bp of Cre-ER^T2 transgene; Cre-R primer binding sites, 976–997 bp of Cre-ER^T2 transgene. The map is drawn in scale. B) Screening SPC-Cre-ER^T2 transgenic mice using PCR. Genomic DNA from each mouse tail was used as template to specifically PCR-amplify the Cre-ER^T2 transgene. M, DNA marker; +, SPC-Cre-ER^T2 plasmid DNA control; -, water control; F8, F13, F16, F42, F67 are five representative founder transgenic mice generated by microinjection of SPC-Cre-ER^T2 expression cassette into fertilized embryos. C) Cre-ER^T2 fusion proteins were detected using Western blot in lung tissues of SPC-Cre-ER^T2 transgenic mice. Lane 1, C57BL/6J mouse; Lane 2, an offspring of F42 founder without Cre-ER^T2 transgene when genotyped using PCR; Lane 3, 4, 5, offspring of F42 founder; Lane 6, 7, 8, offspring of F67 founder. Notice the variable expression levels of Cre-ER^T2 in offspring from the same founder.</p

Tamoxifen-inducible and tissue-specific recombinase activity of Cre-ER^T2 in SPC-Cre-ER^T2/ROSA26R transgenic mice.

<p>A) β-galactosidase activity was detected in the lung alveolar epithelia from a SPC-Cre-ER^T2/ROSA26R transgenic mouse receiving tamoxifen treatment. a & b, lung tissue sections from ROSA26R mice (without Cre-ER^T2 transgene) receiving vehicle (a) or tamoxifen (b); c & d, lung tissue sections from SPC-Cre-ER^T2/ROSA26R transgenic mice receiving vehicle (c) or tamoxifen (d). B) Endogenous β-galactosidase activity was found in lung bronchial epithelia of ROSA26R mouse receiving vehicle (a) and SPC-Cre-ER^T2/ROSA26R transgenic mouse after tamoxifen treatment (b). C) X-gal stained lung tissues of SPC-Cre-ER^T2/ROSA26R transgenic mouse receiving tamoxifen were also immune-stained for proSP-C, an alveolar type II cell-specific marker. Arrows indicate three representative alveolar type II cells co-expressing proSP-C (brown) and β-galactosidase (blue). D) β-galactosidase activity was detected only in the lung alveolar epithelium, but not in other organs from a SPC-Cre-ER^T2/ROSA26R transgenic mouse receiving tamoxifen treatment. a, heart; b, liver; c, kidney; d, intestine; e, lung. All scale bars in this figure equal 100 µm.</p

mTOR was activated during the process of pulmonary fibrosis in vivo and vitro.

<p>A) mTOR activation in fibroblast foci of lung tissue in IPF patients. a,b, H&E staining with normal control and IPF lung tissues; Immunohistochemical staining performed with α-SMA (c,d) and p-S6 (e,f) antibodies showed an increase in α-SMA and p-S6 in IPF lung tissues (d, f) compared with the control (c, e). Scale bar = 100 μm. B) mTOR activation in the lung tissues of C57BL/6J mice after bleomycin intra-tracheal injection. a,b, H&E staining with lungs of saline and bleomycin-treated mice; Immunohistochemical staining was performed with α-SMA (c,d) and p-S6 (e,f) antibodies in saline- and bleomycin-treated mouse lung tissues. NS, normal saline. Scale bar = 100 μm. C) mTOR signaling pathway was activated in primary lung fibroblasts isolated from normal controls treated with TGF-β1(5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated primary lung fibroblasts (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against GAPDH and (c) with p-S6 normalized against S6. **, P<0.01; *, P<0.05. n = 3. D) mTOR signaling pathway was activated in MRC5 cells (a human fetal lung fibroblast cell line) treated with TGF-β1 (5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated MRC5 cells (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against β-actin and (c) with p-S6 normalized against S6. **, p<0.01; *, p<0.05. n = 3.</p

Rapamycin-induced autophagyin the bleomycin-mediated lung injury and fibrosis model.

<p>A) Rapamycin decreased the death caused by bleomycin. Chloroquine, an autophagy inhibitor, reversed the benefit of rapamycin in the bleomycin-mediated lung injury model (Bleo+Rapa+CQ vs Bleo+Rapa, p = 0.0158). B) Western blot analysis of p62 and p-S6 were performed in the bleomycin-mediated lung injury and fibrosis model. p62, a protein inversely correlated with autophagy activity, was decreased in lungs of mice treated with rapamycin alone. p62 expression was higher with combined rapamycin and chloroquine treatment than with rapamycin alone. S6 and β-actin were used as controls. C) Western blot ananlysis of LC3 I and LC3 II were performed in the bleomycin-mediated lung injury and fibrosis mice model. D) Relative density of LC3 II/LC3 I of bands in Fig 5C. Autophagy was significantly decreased in bleomycin-mediated lung injury and fibrosis model (*bleomycin vs normal saline, p < 0.05). E) Electron microscope images of lung tissues show autophagosomes in the bleomycin-mediated lung injury model. Arrows indicate autophagosomes. Rapamycin treatment alone induced an increased number of autophagosomes. Left panel, original magnification: 6,000X and right panel, original magnification: 11,500X. F) Statistical results for the autophagosomes in Fig 5E. The statistical results indicate the percent area of autophagosomes in a cell.</p

Bleomycin-mediated lung injury in wild-type C57BL/6J mice was attenuated by rapamycin (treatment initiated at 5 days before bleomycin injection).

<p>A) H&E staining (a-d) and Masson’s trichrome staining (e-h) of mouse lungs were performed after bleomycin injection at day 21. Lung injury was milder in rapamycin-treated mice (d, h) compared with vehicle-treated mice (c, g). Scale bar = 100 μm. NS, saline; Bleo, bleomycin; Vehi, vehicle; Rapa, rapamycin. B) Semi-quantitative assessment was performed on day 21 using Ashcroft scoring method, a significantly higher score was observed in the mice treated with Bleo (no rapamycin) than those treated with Bleo+Rapa. Results were expressed as mean±SEM, n = 6 mice per group, ** p<0.01. C) Bleomycin-mediated mouse mortality was decreased after rapamycin treatment.</p

Conditional <i>Tsc1</i> knock-down in lung alveolar epithelial cells from doxycyline-treated STT mice exacerbated bleomycin-mediated lung injury.

<p>A) Histological analysis of lungs in the mice treated with bleomycin at day 21. H&E staining (a-c) and Masson’s trichrome staining (d-f) were performed. STT mice had more severe lung injury (c, f) than control mice (b, e). Scale bar = 100 μm. NS, saline; Bleo, bleomycin. B) Semi-quantitative assessment was performed on day 21 using Ashcroft scoring method, a significantly higher score was observed in STT mice treated with Bleo than control mice treated with Bleo. Results were expressed as mean±SEM, n = 6 mice per group, * p<0.05. C) STT mice had a higher mortality rate than control mice after a single intra-tracheal injection of bleomycin for 21 days.</p