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: The sense and antisense primers used for qPCR analysis are: human and mouse S1PR1, sense, 5′-ATC ATG GGC TGG AAC TGC ATC A-3′, antisense, 5′-CGA GTC CTG ACC AAG GAG TAG AT-3; human and mouse S1PR2, sense, 5′-CAG ACG CTA GCC CTG CTC AAG A-3′, antisense, 5′-TAG TGG GCT TTG TAG AGG A-3′; human and mouse S1PR3, sense, 5′-ACA ACC GCA TGT ACT TTT TCA T-3′, antisense, 5′-TAC TGC CCT CCC TGA GGA ACC A-3′; human S1PR4, sense, 5′-GGG CCA TCT TCC GCC TGG TG-3′, antisense, 5′-TGC CCC GCA GGT ACT CCT GG-3′; human S1PR5, sense, 5′-GGC GCG CAC CTG TCC TGT AC-3′, antisense, 5′-TCG GGT CTC TGC CGC AGG AG-3′; human and mouse SphK1, sense, 5′-AAA CCC CTG TGT AGC CTC CC-3′, antisense, 5′-AGC AGG TTC ATG GGT GAC AG-3′; human SphK2, sense, 5′-GCA CAG CAA CAG TGA GCA-3′, antisense, 5′-GAG CCT GAG TGA GTG GGA-3′; porcine TGF-β, sense, 5′-GCA CGT GGA GCT ATA CCA GAA-3′, antisense, 5′-CAT CAA AGG ACA GCC ACT CC-3′; human GAPDH, sense, 5′-GAA GGT GAA GGT CGG AGT-3′, antisense, 5′-GAA GAT GGT GAT GGG TTT C-3′; and mouse GAPDH, sense, 5′-CAC CTT CGA TGC CGG GGC TG-3′, antisense, 5′-GGC CAT GAG GTC CAC CAC CC-3′. cDNA array analysis of mRNA levels of S1PR3 was performed using TissueScan qPCR arrays (HLRT101 and HLRT105, OriGene) following the manufacturer's instructions.

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

Journal: Oncogene

Article Title: B-Myb accelerates colorectal cancer progression through reciprocal feed-forward transactivation of E2F2

doi: 10.1038/s41388-021-01961-9

Figure Lengend Snippet: a Heatmap for B-Myb expression in colorectal cancer. B-Myb expression data were downloaded from the Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/ ). COAD Colon adenocarcinoma, READ rectum adenocarcinoma. b B-Myb mRNA overexpression in colorectal cancer. The TCGA B-Myb expression data were analyzed online by Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/ ). c Verification of B-Myb mRNA overexpression in colorectal cancer. Expression of B-Myb was determined using qRT-PCR with specific primers in colorectal cancer cDNA array purchased from OriGene (HCRT101). Grades: G1 and G2. d B-Myb protein overexpression in colorectal cancer. B-Myb protein expression was determined by immunohistochemical (IHC) analysis using colorectal cancer tissue microarray slides. Representative images (left) and quantification results (right) were indicated. * P < 0.05.

Article Snippet: Expression of B-Myb was determined using qRT-PCR with specific primers in colorectal cancer cDNA array purchased from OriGene (HCRT101).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Immunohistochemical staining, Microarray

Journal: Oncogene

Article Title: B-Myb accelerates colorectal cancer progression through reciprocal feed-forward transactivation of E2F2

doi: 10.1038/s41388-021-01961-9

Figure Lengend Snippet: a Lentivirus-mediated stable B-Myb overexpression. HCT116 and RKO cells were infected with the empty control and B-Myb-expressing lentiviral particles, and then selected in the presence of puromycin to obtain the control (LV-control) and B-Myb overexpression (LV-B-Myb) stable cells. Expression of B-Myb was examined by qRT-PCR and immunoblot analysis. b Lentivirus-mediated stable B-Myb knockdown. HCT116 and RKO cells were infected with the lentiviral particles expressing negative control shRNA and B-Myb shRNA, and then selected in the presence of puromycin to generate the polyclonal control (shNC) and B-Myb knockdown (shB-Myb) stable cells. Expression of B-Myb was examined by qRT-PCR and immunoblot analysis. c , d B-Myb increases cell proliferation. Cell proliferation was detected by CCK8 assay in the stable B-Myb overexpression or knockdown cells at the indicated time points. e , f B-Myb increases colony formation. Cells were seeded on plastic plates for plate clone formation assay. Representative images were shown. g , h B-Myb promotes colorectal cancer growth in vivo. Stable B-Myb overexpression or knockdown HCT116 cells were injected subcutaneously into the dorsal flanks of nude mice. The tumor size was measured 1–2 times a week for tumor growth curve construction. The tumor weight was measured at the end of the experiment. Data represent the mean ± SD. All experiments were performed in triplicates. * p < 0.05, ** p < 0.005, *** p < 0.001.

Article Snippet: Expression of B-Myb was determined using qRT-PCR with specific primers in colorectal cancer cDNA array purchased from OriGene (HCRT101).

Techniques: Over Expression, Infection, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, shRNA, CCK-8 Assay, Tube Formation Assay, In Vivo, Injection

Journal: Oncogene

Article Title: B-Myb accelerates colorectal cancer progression through reciprocal feed-forward transactivation of E2F2

doi: 10.1038/s41388-021-01961-9

Figure Lengend Snippet: a Pearson correlation analysis of B-Myb and E2F2 in colorectal cancer samples ( n = 177). b B-Myb upregulates E2F2 expression. Immunoblotting was performed to determine B-Myb and E2F2 expression in stable B-Myb overexpression and knockdown cells. c Schematic illustration of the wild-type and mutant E2F2-P1314 and B-Myb-P1064 luciferase (Luc) promoter reporter. The transcription start sites for E2F2 and B-Myb genes are indicated as +1. The E2F and Myb-binding sites are shown as boxes. The mutated E2F binding sites (EBS) and/or Myb-binding sites (MBS) are crossed. d Schematic illustration of the wild-type and mutant 3xE2F reporter and luciferase reporter assay. e , f Luciferase reporter assays. HCT116 cells were transiently transfected with the indicated plasmids, and 48 h after transfection, the luciferase activities were measured as described in “Materials and Methods” section. Data are expressed as fold change normalized to the activity of cells transfected with the empty pGL3-basic or pGL4.10 promoter-less vector alone (relative value, 1.0).

Article Snippet: Expression of B-Myb was determined using qRT-PCR with specific primers in colorectal cancer cDNA array purchased from OriGene (HCRT101).

Techniques: Expressing, Western Blot, Over Expression, Mutagenesis, Luciferase, Binding Assay, Reporter Assay, Transfection, Activity Assay, Plasmid Preparation

Journal: Oncogene

Article Title: B-Myb accelerates colorectal cancer progression through reciprocal feed-forward transactivation of E2F2

doi: 10.1038/s41388-021-01961-9

Figure Lengend Snippet: a siRNA mediated silencing of B-Myb and E2F2. Stable B-Myb overexpression (LV-B-Myb, upper panel) HCT116 cells were transiently transfected with negative control siRNA, B-Myb siRNA, or E2F2 siRNA. Stable B-Myb knockdown (shB-Myb, lower panel) HCT116 cells were transiently transfected with pcDNA3.0 empty vector, LV203-B-Myb-Flag and pcDNA3.0-E2F2 expression constructs. Twenty-four hours after transfection, qRT-PCR was performed to determine B-Myb and E2F2 expression. b E2F2 is required for B-Myb-induced cell proliferation. Cells were transiently transfected as described in ( a ), and cell proliferation was detected by CCK8 assay at the indicated time points. c GSEA plot showing that the B-Myb-regulated genes correlate with CSR_LATE gene signatures (CSR_LATE_UP.V1_UP). d B-Myb is essential to activation of ERK and AKT pathways. Immunoblotting was performed to determine B-Myb, ERK, p-ERK, and p-AKT expression in the stable B-Myb overexpression (LV-B-Myb) and its control (LV-vector) HCT116 cells (Left). Stable B-Myb knockdown (shB-Myb) and its control (shNC) HCT116 cells were subjected to serum starvation for 2 h, and then treated with EGF (200 ng/mL) for 15 min. Immunoblotting was performed to determine p-ERK expression (Right). e E2F2 is required for B-Myb-induced activation of ERK and AKT pathways. Stable B-Myb overexpression (LV-B-Myb) HCT116 and RKO cells were transiently transfected with negative control (NC) siRNA, B-Myb siRNA and E2F2 siRNA, respectively. Forty-eight hours later, cells were subjected to immunoblotting with the indicated antibodies. The band intensity ratios of pAKT or pERK to GAPDH are shown below the bands. f B-Myb and E2F2 activate ERK and AKT pathways. Stable B-Myb knockdown (shB-Myb) HCT116 and RKO cells were transfected with pcDNA3.0 empty vector, LV203-B-Myb-Flag and pcDNA3.0-E2F2 expression constructs, respectively. Forty-eight hours later, cells were subjected to immunoblotting with the indicated antibodies. g Work model. B-Myb and E2F2 regulate the transcription of each other (reciprocal feed-forward regulation) as well as their own transcription (autoregulation). In addition, B-Myb and E2F2 associate with each other (collaboration) and regulate various downstream target genes to activate ERK and AKT pathways and induce the malignant cell phenotype in colorectal cancer. Data represent the mean ± SD. All experiments were performed in triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Expression of B-Myb was determined using qRT-PCR with specific primers in colorectal cancer cDNA array purchased from OriGene (HCRT101).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, Expressing, Construct, Quantitative RT-PCR, CCK-8 Assay, Activation Assay, Western Blot

Journal: Oncogene

Article Title: The Hedgehog processing pathway is required for NSCLC growth and survival

doi: 10.1038/onc.2012.243

Figure Lengend Snippet: a, SKN and b, DISP-1 are differentially expressed in NSCLCs as compared to the corresponding matched normal lung tissue. Expression levels from 58 matched NSCLC/normal lung tissue pairs were assessed by oligonucleotide micro-array analyses. Gene expression in NSCLC was normalized to matched normal tissue and plotted as the Log10 change (tumor/normal). c, SKN is overexpressed in commercially available clinical lung cancer specimens, confirming the pattern of expression detected by micro-array analysis. These were purchased from Origene (Tissuescan Lung Cancer cDNA Array IV) and consist of 24 matched lung cancer/normal lung cDNA pairs pre-normalized to β-ACTIN. SKN expression was determined by qRT-PCR, and lung tumor expression was normalized to matched normal control. d, DISP-1 is differentially expressed in clinical lung cancer specimens. The same Tissuescan cDNA array was used to assess DISP-1 levels by qRT-PCR. Arrows represent pairs where DISP-1 expression was below the threshold of detection in either normal lung tissue (up arrow) or lung tumor (down arrow). e, Higher DISP-1 expression is associated with reduced recurrence free survival. A publically available dataset (GSE8894) consisting of micro-array profiles of 138 NSCLC cases and an associated clinical survival database (recurrence-free survival) was mined to find correlations between DISP-1 expression and recurrence free survival. Lung cancers were separated into tertiles based on DISP-1 expression, and the top third compared to the bottom third. f, Higher DISP-1 expression is associated with reduced survival in a second publically available dataset (GSE10245) consisting of micro-array profiles of 58 NSCLC cases and an associated clinical outcome database (overall and progression-free survival). This dataset was analyzed in the same manner as for GSE10245. P-values reported are from a one-tailed Log-rank (Mantel-Cox) test.

Article Snippet: These were purchased from Origene (Tissuescan Lung Cancer cDNA Array IV) and consist of 24 matched lung cancer/normal lung cDNA pairs pre-normalized to β- ACTIN .

Techniques: Expressing, Microarray, Quantitative RT-PCR, One-tailed Test

Journal: Oncogenesis

Article Title: Ultraconserved region-containing Transformer 2β4 controls senescence of colon cancer cells

doi: 10.1038/oncsis.2016.18

Figure Lengend Snippet: Expression of TRA2β4 in colon cancers. ( a ) HCT116 cells were transfected with pcDNA3.1 (mock), pCMV- TRA2β4 or pCMV- TRA2β4 mutated in the stem-loop motif (pCMV- TRA2β4mt , 485- AA GG-488) for 48 h. mRNA levels were measured by qPCR using GAPDH as an endogenous quantity control. *Significantly decreased compared with mock-treated cells ( P <0.05 by Student's t -test). ( b ) After transfection of HCT116 cells (1 × 10 4 cells per 24-well dish) with pcDNA3.1 (mock), pCMV- TRA2β4 or pCMV- TRA2β4mt , the number of growing cells were measured using CellTiter96 AQueous Cell Proliferation Assay (MTS). Values expressed as means±s.d. ( n =3). *Significantly increased compared with mock-transfected cells ( P <0.05 by Student's t -test). ( c ) Using human colon cancer tissue qPCR arrays (TissueScan, HCRT103), TRA2β4 expressed in cDNAs from adenocarcinomas of the colon and surrounding normal colon tissues were measured by qPCR in 24 patients. Values were normalized to ACTB mRNA levels.

Article Snippet: TissueScan Tissue qPCR Arrays (HCRT103) including cDNAs from paired normal and tumor tissues in 24 patients with adenocarcinomas of the colon were obtained from OriGene Technologies (Rockville, MD, USA), and TRA2β4 levels in normal and tumor tissues were determined by qPCR.

Techniques: Expressing, Transfection, Proliferation 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: 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

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: Oncotarget

Article Title: Targeting MCT-1 oncogene inhibits Shc pathway and xenograft tumorigenicity

doi:

Figure Lengend Snippet: MCT-1 mRNA expression levels in human lung cancers The TissueScan lung cancer tissue cDNA arrays Panel II, III and V consisted of a total of 19 normal lung samples and 124 lung cancer biopsies from different individuals were analyzed the expression of MCT-1 mRNA by Q-RT-PCR. The MCT-1 mRNA level in each tumor sample was normalized to β-actin mRNA and calibrated to the overall mean of MCT-1 mRNA level of normal tissue (set as 1-fold). MCT-1 mRNA had a >2-fold induction in tumor samples over normal lung tissue were defined as the gene high-activation. The statistical analysis used Fisher’s exact test.

Article Snippet: The TissueScan Lung Cancer Tissue qPCR Array (Panel II, III and V) (OriGene Technologies, Inc.,) was analyzed the level of MCT-1 mRNA expressed in human lung carcinomas, in which the MCT-1 mRNA revealed a 2-fold induction over the mean of normal lung tissue were recognized as high expression of MCT-1 gene.

Techniques: Expressing

Journal: Oncotarget

Article Title: Targeting MCT-1 oncogene inhibits Shc pathway and xenograft tumorigenicity

doi:

Figure Lengend Snippet: Shc mRNA expression levels in human lung cancers The TissueScan lung cancer tissue cDNA arrays (Panels II, III and V) were used to analyze the Shc gene (three isoforms) expression by Q-RT-PCR analysis. The Shc mRNA level identified in tumors was normalized to β-actin and calibrated to the overall mean of Shc mRNA level of normal lung tissue. Shc mRNA levels in tumors which elevated a 1.5- fold increase over normal breast tissues were defined as the high-activation of Shc gene. Shc transcripts were observed to be induced in lung cancers. The statistical analysis used Fisher’s exact test.

Article Snippet: The TissueScan Lung Cancer Tissue qPCR Array (Panel II, III and V) (OriGene Technologies, Inc.,) was analyzed the level of MCT-1 mRNA expressed in human lung carcinomas, in which the MCT-1 mRNA revealed a 2-fold induction over the mean of normal lung tissue were recognized as high expression of MCT-1 gene.

Techniques: Expressing