1. Intracellular TGF-β Receptor Blockade Abrogates Smad-Dependent Fibroblast Activation In Vitro and In Vivo 
Wataru Ishida, Yasuji Mori, Gabriella Lakos, Lihong Sun, Feng Shan, Scott Bowes, Serene Josiah, Wen-Cherng Lee, Juswinder Singh, Leona E. Ling, John Varga 
Journal of Investigative Dermatology 
Volume 126, Issue 8, Pages (August 2006)
DOI: /sj.jid
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
SM305 Ligand displacement assay. SM305 was tested for its ability to displace ligand occupation of the ATP-binding site. Indicated concentrations of SM305 were incubated with recombinant human ALK5 kinase and the radiolabeled ATP-competitive ALK5 inhibitor HTS Dose-dependent displacement of the radioligand was observed and the Ki of SM305 was determined to be 0.8nM.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Modulation of intracellular TGF-β signaling. Confluent foreskin fibroblasts were incubated with DMSO or SM305 (10μM or indicated concentrations), followed 60minutes later by TGF-β1 (2.5ng/ml). (a) After indicated periods of incubation, cultures were harvested, and whole cell lysates were subjected to Western analysis, or (b) immunoprecipitated with antibodies to Smad1/2/3 or IgG, followed by Western analysis. Lower panel, Western blot of whole cell lysates. Immunoblots representative of several independent experiments are shown. (c) Cytotoxicity was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based assay. The values shown represent the means±SD of the proportion of viable cells from triplicate determinations.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Modulation of Smad nuclear function. Confluent foreskin fibroblasts were incubated with DMSO, SM305, or TGF-β1 (2.5ng/ml) for 90minutes. (a–e) SM305 was added 30minutes before (b and d), or (e) 30minutes after TGF-β1. At the end of the incubation, fibroblasts were fixed, stained with anti-Smad2/3 antibodies, and observed by confocal microscopy. 4,6-Diamidino-2-phenylindone identifies nuclei. Representative images are shown (original magnification × 250). Values in parentheses indicate the percent of fibroblasts showing predominantly nuclear Smad2/3. (f) Nuclear extracts were prepared and incubated with biotinylated SBE oligonucleotides. Protein–DNA complexes were precipitated and examined by DAPA using antibodies to phospho-Smad2 or phospho-Smad3, as described under Materials and Methods. A representative image is shown.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Modulation of fibrotic TGF-β responses by SM305. Fibroblast cultures were incubated with SM305 (10μM) 30minutes before, or 8 or 24hours after TGF-β (12.5ng/ml). Following further 48hours incubation, cultures were harvested. (a) Total RNA was isolated and examined by Northern analysis. Representative autoradiograms are shown. (b) Concentrations of TGF-β1 and IL-6 in the supernatants were determined by ELISA. The values are expressed as means±SD from triplicate determinations. (c–g) Fibroblasts were incubated with TGF-β1 (2.5ng/ml) with or without SM305 for 5 days. SM305 was added (c-f) 30minutes before, or (g) 30minutes after TGF-β1. At the end of the incubation, slides were stained with antibodies to α-smooth muscle actin (green color) or with rhodamine (red color), and observed by confocal microscopy. 4,6-Diamidino-2-phenylindone identifies nuclei. Representative images are shown (original magnification × 250). (h) Total RNA was examined by Northern analysis. A representative Northern blot is shown.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Effects of SM305 to Smad-dependent transcriptional responses. Subconfluent fibroblasts were transiently transfected with pSBE4-Luc along with an internal control, and following pretreatment with indicated concentrations of SM305, were incubated with TGF-β1 (12.5ng/ml) for 24hours. Cultures were harvested, and whole cell lysates assayed for luciferase activities. The results are shown as means±SD relative to controls from triplicate determinations from a representative experiment.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
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7. Figure 6
The effect of SM305 in scleroderma fibroblasts. Confluent cultures of scleroderma (three individual cell lines) and normal skin fibroblasts (two cell lines) were incubated in parallel with SM305 (10μM) for 90minutes in the absence (scleroderma) or presence (normal) of TGF-β (2.5ng/ml). At the end of the incubation, fibroblasts were fixed, stained with antibodies to (a) Smad2/3 or (b) to α-smooth muscle actin, and observed by confocal microscopy. Representative images are shown; original magnification × 250; inset, higher magnification. (c) Left panel, the relative levels of nuclear Smads were determined by quantitative analysis of the confocal microscopy data to determine the two-color correlation coefficient with Zeiss LSM510 software. Open bars, control fibroblasts; closed bars, scleroderma fibroblasts. Right panel, the number of smooth muscle actin-positive fibroblasts was determined by quantitative image analysis. The values (means±SEM from multiple fields from three independent experiments) represent the proportion of fibroblasts displaying positive staining. Open boxes, control fibroblasts; closed boxes, scleroderma fibroblasts. (d) Confluent cultures of fibroblasts from five patients with scleroderma (S1–S5) were incubated with SM305 (bar=10μM) for 24hours, and whole cell lysates were examined by Western analysis. Signal intensities for type I collagen were quantitated by densitometry, and the results normalized for vimentin in the same lanes are shown under the autoradiograms.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
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8. Figure 7
SM305 abrogates TGF-β-induced Smad activation in dermal fibroblast in vivo. Transgenic mice harboring 17kb of the mouse COL1A2 promoter fused to luciferase gene were injected with PBS, TGF-β1 (100ng), or SM305 (20nM) singly or in combination. Mice were killed 24hours later and full thickness samples of skin were examined as described in Materials and Methods. (a) Phospho-Smad2/3 expression by immunohistochemistry. Fibroblasts in TGF-β1-injected skin showed markedly enhanced phospho-Smad2/3 expression (bar=50μm). Note strong nuclear phospho-Smad2/3 staining (Original magnification × 1,000, bar=25μm). Right upper quadrant, proportion of fibroblasts positive for phospho-Smad2/3. The values represent the means±SD from two independent experiments. (b) Sections were stained with hematoxylin and eosin (bar=100μm); Inset, original magnification × 400 (bar=25μm). Right upper quadrant, quantitation of infiltrating cells. Values are the means±SD from quadruplate determinations.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
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9. Figure 8
SM305 abrogates TGF-β-induced COL1A2 promoter activity in vivo. (a) Dermal fibroblasts were established from newborn transgenic pups or wild-type littermates and were studied at second passage. Confluent cultures were incubated with SM305, followed by TGF-β for 48hours. At the end of the incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. Luciferase activity in wild-type fibroblasts is undetectable. Results are the means±SD for triplicate samples from a representative experiment. (b) Transgenic mice were injected with PBS, TGF-β1 (100ng), or SM305 (20nM) singly or in combination. Mice were sacrificed 24hours later, cell lysates were prepared from full thickness injection site skin, and assayed for luciferase activity. The values represent the means±SD of luciferase activities relative to PBS (3–5mice/group). * indicate P<0.05.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions