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

Journal: Cell Death & Disease

Article Title: Human BCL-G regulates secretion of inflammatory chemokines but is dispensable for induction of apoptosis by IFN-γ and TNF-α in intestinal epithelial cells

doi: 10.1038/s41419-020-2263-0

Figure Lengend Snippet: a Relative mRNA expression of BCL-G S/L measured by RT-qPCR in the indicated adult human tissues. Relative mRNA expression of b BCL-G S and c BCL-G L measured by RT-qPCR in colonic biopsy tissues isolated from non-IBD individuals ( n = 20), patients with inactive ( n = 20) or active ( n = 24) ulcerative colitis, and patients with inactive ( n = 19) or active ( n = 21) Crohn’s disease. BCL-G S/L expression data in active ulcerative colitis were used for correlation analysis in Fig. . For panels ( b ) and ( c ), data shown include the median with interquartile range. d Relative mRNA expression of BCL-G S/L measured by RT-qPCR in TissueScan cDNA array of 24 matched samples (normal, uninvolved colon vs. colorectal cancer) covering clinical stage I ( n = 5), II ( n = 7), III ( n = 8) and IV ( n = 4). # p < 0.05, ### p < 0.001 (Kruskal–Wallis test followed by Dunn’s multiple comparisons test as indicated), $ p < 0.05, $$$ p < 0.001 (repeated measures two-way ANOVA followed by Fisher’s LSD test as indicated), ‡‡ p < 0.01, ‡‡‡ p < 0.001 (repeated measures two-way ANOVA followed by Fisher’s LSD test vs. normal colon, stage I). IBD — inflammatory bowel disease, UC — ulcerative colitis, CD — Crohn’s disease.

Article Snippet: To study cancer-associated gene expression, Colon Cancer TissueScan cDNA array III (cat# HCRT303, OriGene) was used.

Techniques: Expressing, Quantitative RT-PCR, Isolation

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.

Article Snippet: To verify whether or not RecQL4 expression is elevated in human breast tumor tissue samples, RecQL4 mRNA level was examined using TissueScan Human Breast cancer tissue qPCR array (Origene, Rockville, MD 20850).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: FXR Controls the Tumor Suppressor NDRG2 and FXR Agonists Reduce Liver Tumor Growth and Metastasis in an Orthotopic Mouse Xenograft Model

doi: 10.1371/journal.pone.0043044

Figure Lengend Snippet: A , Ndrg2 mRNA induction in livers of C57Bl6 mice by FXR agonists. C57Bl6 mice on normal chow received a bolus of vehicle (n = 9), or 30 mg/kg of Px20606 or 30 mg/kg of GW4064 in vehicle (n = 6 each) by oral gavage. Mice were sacrificed after 4 h and liver RNA isolated for RT-qPCR analysis. Ndrg2 and Shp mRNA expression levels were calculated according to the ΔΔCt method using normalization to Tbp as a house keeping transcript and the mean level of Ndrg2 and Shp expression in vehicle treated mice were set to 1.0. Data are presented as the mean ± standard error of the mean (±SEM). Statistical significance in a nonpaired Student's t-test: *, ** and *** = p<0.05, <0.01 and <0.001. B , Quantitative determination of mRNA's in livers of wild type (wt) and FXK −/− (KO) mice. Relative mRNA levels of Ndrg2, Shp, Cyp7a1 and Cyclophilin E were determined by RT-qPCR from wt (n = 5) and FXR −/− (n = 5) mice as indicated. Tbp-normalized relative mRNA levels of wt mice were set to 1.0 respectively. C , Decreased expression of FXR and the FXR target genes NDRG2 and SHP in human HCC. FXR, NDRG2 and SHP mRNA levels were quantified by RT- qPCR in 8 normal, 34 HCC from different stages (7 samples stage I, 8 samples of stages II and IIIA, 3 samples of stage IV) and 12 non-HCC liver disease (LD) derived cDNA's. Relative folds of mRNA expression levels between normal livers and the respective stages of HCC or non-HCC liver disease were calculated according to the ΔΔCt method using normalization to TBP. The mean levels of FXR, NDRG2 and SHP expression in normal livers were set to 1.0 respectively and relative mRNA levels are expressed as mean ± SEM. Statistical significance in a nonpaired Student's t-test: *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: The HCC samples included seven samples of stage I HCC, eight samples of stage II and IIIA HCC each, and three samples of stage IV HCC and 13 samples of non HCC liver diseases ( http://www.origene.com/assets/documents/TissueScan/LVRT101.xls ;).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Derivative Assay