Fig. 1. Bronchoalveolar Macrophages Stimulate hSP-B 1 Fig. 1. Bronchoalveolar Macrophages Stimulate hSP-B 1.5-kb lacZ Reporter Gene Expression in AT II Epithelial Cells AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and cocultured with bronchoalveolar macrophages (MC) isolated from BALF. Cocultured cells were treated with or without LPS (10 ng/ml). Control represents AT II epithelial cells without macrophage coculturing. Cells were harvested for measurement of β-galactosidase activities. Values are means ± sd; n = 3 (mice). From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 2. Heat Treatment Abolishes Stimulation of the hSP-B 1 Fig. 2. Heat Treatment Abolishes Stimulation of the hSP-B 1.5-kb lacZ Reporter Gene by Bronchoalveolar Macrophage Medium AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and incubated with heat-treated or untreated bronchoalveolar macrophage (MC) mediums (LPS treated or untreated). Control represents AT II epithelial cells without addition of macrophage medium. Cells were harvested for measurement of β-galactosidase activities. Values are means ± sd; n = 3 (mice). From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 3. IL-6 Ab Inhibits Bronchoalveolar Macrophage Stimulation of hSP-B 1.5-kb lacZ Reporter Gene Expression in AT II Epithelial Cells AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and cocultured with bronchoalveolar macrophages (MC) with or without LPS treatment (10 ng/ml). IL-6 Ab (5 μl) was added to cocultured cells. Cocultured cells without IL-6 Ab addition served as comparison. Control represents AT II epithelial cells without macrophage coculturing. Cells were harvested for measurement of β-galactosidase activities. Values are means ± sd; n = 3. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 4. Expression of IL-6 Receptor Subunits and Phosphorylated Stat3 Y705 in Respiratory Epithelial Cells A, Western blot analysis of IL-6 receptor subunits in AT II epithelial cells. AT II epithelial cells were isolated from wild-type FVB/N mouse lungs. Protein extracts were prepared for SDS denaturing gel electrophoresis. Western blot analyses were performed using antibodies against IL-6R or gp130 subunits. B, Immunohistochemical staining of phosphorylated Stat3 Y705 in the lung. Lung sections from IL-6 treated (IL-6) or untreated (PBS) adult FVB/N mice were stained with an antibody specifically against phosphorylated Stat3 at Y705. More positively stained Clara cells or alveolar epithelial cells (some stained cells are represented by arrows) were detected in the IL-6-treated lungs. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 5. Functional Domain Swapping between Stat3 and Stat1 in Stimulation of hSP-B 500 Luciferase Reporter Gene in H441 Cells A, Illustration of functional domain swapping between Stat3 and Stat1. B, Stimulation of the hSP-B 500 luciferase reporter gene by Stat3, Stat1, and various Stat3/Stat1 hybrids in H441 cells. Stimulation of the hSP-B 500 luciferase reporter gene without cotransfection was used as control. Values are means ± sd; n = 3. S1, Stat1; S3, Stat3; ND, N-terminal domain; CC, coiled coil domain; DB, DNA-binding domain; SH2, SH2 domain; Y701, Y701 domain; TAD, transcription activation domain From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 6. Interaction between the Stat3 SH2 Domain and RARα in GST Pull-Down Study A, Pull down of full-length RARα by Stat3 SH2 GST fusion protein. No enhanced pull-down signal of full-length TTF-1 was observed by Stat3 SH2 GST fusion protein. B, Pull down of the RARα LBD domain by Stat3 SH2 GST fusion protein. AF-1, RARα ligand-independent activation domain; DBD, RARα DNA-binding domain; LBD, RARα ligand-binding domain; AF-2, RARα ligand-dependent activation domain. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 7. Synergy of DNA Binding and Transactivation between Stat3 and RARα/RXRγ A, Stimulation of the hSP-B 500 luciferase reporter gene by Stat3, RARα/RXRγ, or combination in H441 cells. Stimulation of the hSP-B 500 luciferase reporter gene without cotransfection was used as control. Values are means ± sd; n = 3. B, H441 cells were transfected with expression vectors of Stat3, RARα/RXRγ, or combination. Soluble chromatin was immunoprecipitated with Stat3 antibody. Coprecipitated DNA was analyzed by PCR using a pair of primers corresponding to the enhancer region (−500 to −331 bp) of the hSP-B 500 5′-flanking regulatory sequence. The hSP-B 500 luciferase reporter gene construct was used as positive control. No DNA was added in negative control. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 8. Generation of Doxycycline-Controlled dnStat3 Transgenic Mice A, Illustration of constructs for generating SP-C-rtTA/(Teto)7-CMV-dnStat3 double-transgenic mice. B, Expression of dnStat3 mRNA, SP-B mRNA, and GAPDH mRNA in SP-C-rtTA/(Teto)7-CMV-dnStat3 double-transgenic mice. Total RNAs isolated from triplicate doxycycline-treated or untreated double-transgenic mice were used for RT-PCR analysis using a pair of specific primers for the (Teto)7-CMV-dnStat3 DNA sequence, the SP-B cDNA sequence, or the GAPDH cDNA sequence. Dox, Doxycycline. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 9. Morphometrical Analysis of SP-C-rtTA/(Teto)7-CMV-dnStat3 Double-Transgenic Mouse A, Disruption of the alveolar structure in the adult lung of the SP-C-rtTA/(Teto)-CMV-dnStat3 FVB/N double-transgenic mice after doxycycline treatment. The lung sections were stained with hematoxylin and eosin. The doxycycline-treated wild-type mice were used as control. WT, Wild-type mice; dnStat3, double transgenic mice. B, Ratio increase of the pulmonary airspace vs. parenchyma in double-transgenic mice after dnStat3 overexpression. Values are means ± sd; n = 3–5. ANOVA showed significant differences between wild-type and double-transgenic mice, P < 0.05. Wt, Wild-type mice; Tg, double-transgenic mice. C, Frequency distribution of the two-dimensional alveolar area in wild-type and double-transgenic mice after dnStat3 overexpression. wt, Wild-type mice; Tg, double transgenic mice From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society

Fig. 10. Survival of Doxycycline-Treated Mice under Hyperoxic Condition Doxycycline-treated adult wild-type and SP-C-rtTA/(teto)₇-CMV-dnStat3 double-transgenic mice were exposed to 95% O₂. Animal mortality was recorded every 12 h. x-axis, Days exposed to 95% O₂; y-axis, survival percentage of animals. The solid line represents the survival rate of wild-type animals. The dashed line represents double-transgenic mice. In each group, n = 20. Logrank test showed significant difference between survival rates of two groups. WT, Wild-type mice; dnStat3, SP-C-rtTA/(teto)₇-CMV-dnStat3 double transgenic mice. From: Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-α in Regulation of the Surfactant Protein B Gene in the Lung Mol Endocrinol. 2004;18(6):1520-1532. doi:10.1210/me.2003-0458 Mol Endocrinol | Copyright © 2004 by The Endocrine Society