Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: Up-regulation of S1PR3 in human lung adenocarcinomas. A, qPCR quantitation of S1PR3 mRNA in cDNA arrays of human lung adenocarcinoma specimens (OriGene, HLRT101 and HLRT105). **, p < 0.01, Student's t test. B, qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105). **, p < 0.01, Student's t test. C, HEK293 cells were transfected with S1PR3 or pcDNA vector. Transfected cells were immunostained with anti-S1PR3 (Cayman Chemical) (IMF, left panels). Arrows, nonspecific fluorescent precipitates used for image orientation. Scale bar = 33 μm. D, anti-S1PR3 staining of human lung adenocarcinoma tumor microarray (Accumax 306). AdC, adenocarcinoma; N, adjacent normal lung tissue. E, immunostaining intensity was quantitated with the National Institutes of Health ImageJ software. Data, analyzed with GraphPad Prism 5 software, are shown as mean ± S.E. Statistical significance was analyzed by Student's t test. F, representative images of anti-S1PR3 staining of human lung adenocarcinoma and the respective adjacent normal lung epithelial tissue. G, quantitation of anti-S1PR3 staining of human lung squamous carcinoma microarray (Accumax 306). Data are mean ± S.E. Statistical significance was analyzed by Student's t test. H, representative images of anti-S1PR3 staining of human lung squamous carcinoma and the respective adjacent normal lung epithelial tissue.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Quantitation Assay, Transfection, Plasmid Preparation, Staining, Microarray, Immunostaining, Software

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: TGF-β/SMAD3 signaling axis up-regulates S1PR3. A, HBEC2-KT cells were treated with TGF-β (1 ng/ml) for various times. mRNA levels of S1P receptors were measured by qPCR analysis. Data are mean ± S.D. of triplicate determinations. *, p < 0.05, Student's t test. B, protein levels of S1PR3 in TGF-β (1 ng/ml)-treated HBEC2-KT cells. Lower panel, Western blot intensity was quantitated by National Institutes of Health ImageJ. Data (normalized to actin) are mean ± S.D. of triplicate determinations. * and **, p < 0.05 and 0.01, respectively, Student's t test. C, CHO cells were transduced with adenoviral particles (multiplicity of infection of 200) carrying S1PR1, S1PR2, or S1PR3 vector for 20 h as we described (8). Extracts were blotted with antibody against S1PR3 (Cayman), S1PR2 (Cayman), or S1PR1 (E49) (8). D, mRNAs of S1PR3 and TGF-β in minced C57BL/6 mouse lungs (∼1–2 mm3) infected with adenoviral active TGF-β (Ad-TGF-β, 1 × 108 pfu/ml) or empty vector (Ad-Ctrl) (37 °C, 24 h). **, p < 0.01, n = 5, Student's t test. E, mRNA levels of SphK1 and SphK2 in TGF-β-treated HBEC2-KT cells. F, HBEC2-KT cells (2 × 106 cells in 100-mm dish, 10 ml of cultural medium) were treated with TGF-β (1 ng/ml) for 24 h. Medium was quantitated for S1P, ceramide (Cer), and sphingomyelin (SPM) by LC-MS/MS (29, 46). G, HBEC2-KT were pretreated for 30 min with inhibitors. S1PR3 levels were measured by qPCR, following TGF-β treatment (4 h). The following inhibitors were used: SB4, TGF-β receptor I (SB-431542, 10 μm); SIS3, SMAD3 (2 μm); SB2, p38 kinase (SB-203580, 50 nm); BAY, NFκB (BAY11-7085, 10 μm); JII, JNK (JNK inhibitor II, 10 μm). *, p < 0.05; dashed line, non-statistical significance; n = 3, ANOVA. Each experiment was repeated 2–3 times with similar results. H, cells were pretreated for 30 min with inhibitors, followed by stimulation with TGF-β (1 ng/ml). Activation of p38, JNK, and NFκB was measured by Western blotting with phospho-p38 (P-p38), phospho-JNK (P-p54JNK and P-p46JNK), and phospho-IκBα (p-IκBα). Inhibitors used are: SB-203580 (50 nm) for p38 kinase, JNK inhibitor II (10 μm) for JNK, and BAY11-7085 (10 μm) for NFκB.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Western Blot, Transduction, Infection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: S1PR3 regulates growth and lung colonization of lung adenocarcinoma cells. A, H1793 cells were stably transfected with sh-S1PR3 or pRS (sh-Ctrl) vector (11, 16). mRNA levels of S1PR3 were quantitated with qPCR analysis. B, H1793 cells (1 × 106 cells), stably transfected with sh-S1PR3 or sh-Ctrl vector, were subcutaneously inoculated in Scid mice. Tumor volume was measured in two dimensions using calipers, and volume was determined using the formula width2 × length × 0.52 (49). C, 4 weeks after inoculation, tumors were removed and weighed. D, Scid mice were injected with H1793 cells transfected with sh-S1PR3 or sh-Ctrl vector (1 × 106 cells) via tail vein route. 28 days later, tumor nodules on lung surface were scored. E, representative images of lung injected with H1793-sh-Ctrl and H1793-sh-S1PR3 cells. Arrows, tumor nodules. Scale bar = 0.5 cm. F, volume of xenograft tumors in athymic nude mice subcutaneously implanted with H1299 cells stably transfected with S1PR3 or control pcDNA vector (1 × 106 cells) (11, 16). G, qPCR quantitation of S1PR3 levels in H1299/pcDNA and H1299/S1PR3 cells. **, p < 0.01, n = 6, ANOVA.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Injection, Quantitation Assay