Journal: International Journal of Biological Sciences

Article Title: ETV4 interacts with LOXL2 to induce epigenetic activation of NID1 during colorectal cancer progression

doi: 10.7150/ijbs.116383

Figure Lengend Snippet: Upregulation of ETV4 is related with progression of CRC. a. Venn diagram of differential expression genes (DEGs) overlapped among the three datasets ( GSE4183 , GSE20916 and TCGA-COAD). b. The complete PPI network of common DEGs in CRC. Each circle represents one gene, and each line indicates one protein-protein interaction. Figure inside the circle indicates the corresponding protein structure. c. ETV4 mRNA overexpression in CRC. The TCGA ETV4 expression data were analyzed online by GEPIA. (COAD, Colon adenocarcinoma; READ, Rectum adenocarcinoma). d. Expression of ETV4 mRNA was detected via RT-qPCR with specific primers in CRC cDNA array purchased from OriGene. e-f. The correlation of ETV4 mRNA expression with lymphatic metastasis (p<0.001) or pathologic grade (p<0.001) was analyzed by RT-qPCR using CRC cDNA array. g. ETV4 protein expression was verified by immunohistochemical (IHC) assay using CRC tissue microarray slides (p<0.001), scale bar: 25μm. h-i. The association of ETV4 protein expression with lymphatic metastasis (p<0.01) or pathologic grade (p<0.01) was analyzed by immunohistochemistry and quantitative immunohistochemistry analysis, scale bar: 25μm. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: To confirm the above bioinformatics results, the human CRC cDNA array from Origene was used to detect the ETV4 mRNA expression, and the results showed that ETV4 was indeed remarkably elevated in CRC samples compared with the normal colorectal tissues (p<0.001, Fig. d), and of note, the expression level of ETV4 mRNA was positively correlated with lymphatic metastasis (p<0.001, Fig. e) and pathologic grades (p<0.001, Fig. f).

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

Journal: International Journal of Biological Sciences

Article Title: ETV4 interacts with LOXL2 to induce epigenetic activation of NID1 during colorectal cancer progression

doi: 10.7150/ijbs.116383

Figure Lengend Snippet: LOXL2 is a new target gene of ETV4. a. Verification of ETV4-regulated downstream genes which were involved in EMT in stable ETV4 overexpression HCT116 cells. b. Determination of ETV4-regulated downstream genes which were involved in EMT by RT-qPCR in ETV4 knockdown HCT116 cells. c. Confirmation of correlation between ETV4 mRNA and LOXL2 mRNA via GSE4183 and GSE20916 . d. Verification of the relation of ETV4 and LOXL2 by RT-qPCR using the human CRC cDNA array. e. Determination of association between EMT markers and ETV4 or LOXL2 via GSE4183 and GSE20916 . The size and color intensity of the circles encode the correlation magnitude: larger circles with darker hues indicate stronger correlations, while smaller, lighter circles represent weaker associations. f. Immunoblotting was conducted to determine ETV4 and LOXL2 expression in stable ETV4 overexpression and knockdown HCT116 cells. g. Schematic illustration of the wildtype and mutant LOXL2-P1773 luciferase (Luc) promoter reporters. The transcription site for LOXL2 gene is indicated as +1. The ETV4 binding sites are shown as boxes. The mutated sites are crossed. h. ChIP assay. Chromatin fragments were prepared from HCT116 cells and immunoprecipitated with anti-ETV4 antibody or control IgG. The precipitated DNA was then amplified by real-time PCR with primers directed to the ETV4 binding sites in the LOXL2 promoter region. i. Luciferase reporter assays. 293Ta cells were transiently transfected with the indicated plasmids, and forty-eight hours after transfection, the luciferase activities were measured.

Article Snippet: To confirm the above bioinformatics results, the human CRC cDNA array from Origene was used to detect the ETV4 mRNA expression, and the results showed that ETV4 was indeed remarkably elevated in CRC samples compared with the normal colorectal tissues (p<0.001, Fig. d), and of note, the expression level of ETV4 mRNA was positively correlated with lymphatic metastasis (p<0.001, Fig. e) and pathologic grades (p<0.001, Fig. f).

Techniques: Over Expression, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Mutagenesis, Luciferase, Binding Assay, Immunoprecipitation, Control, Amplification, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Biological Sciences

Article Title: ETV4 interacts with LOXL2 to induce epigenetic activation of NID1 during colorectal cancer progression

doi: 10.7150/ijbs.116383

Figure Lengend Snippet: NIDI is a downstream gene of ETV4/LOXL2-induced aggressive phenotype in CRC. a. Venn diagram showing the candidate genes of ETV4 and LOXL2. The genes of which the Pearson correlation coefficient with LOXL2 was greater than 0.7, and combined with the differential expression genes (|log2FC| ≥ 2.0) in our previous ETV4 overexpression RNA-seq data, finally yielding 79 candidate genes. b-c . Expression of NID1 was determined by RT-qPCR (b) and immunoblot analysis (c) in HCT116 and RKO cells with stable ETV4 overexpression. d-e. Expression of NID1 was determined by immunoblot analysis (d) and RT-qPCR (e) when silencing LOXL2 in HCT116 and RKO cells with stable ETV4 overexpression. f. Expression of ETV4, LOXL2 and NID1 in paired non-tumor and tumor tissues (n=4) was determined by immunoblot analysis. N, normal tissue; C, cancer tissue. g. The correlation of ETV4 or LOXL2 with NID1 was detected via RT-qPCR in CRC cDNA array purchased from OriGene. h. Stable ETV4 overexpression HCT116 cells were transiently transfected with negative siRNA or NID1 siRNA. The cell viability was determined by CCK8 assay at the indicated time points. i. Stable ETV4 knockdown HCT116 cells were transiently transfected with empty or pcDNA3.0-NID1. The cell viability was determined by CCK8 assay at the indicated time points. j-k. HCT116 cells were treated as described in (h) or (i) for 24h, then cells were subjected to transwell migration and invasion assay (i, scale bar: 100μm). Data represent the mean ± SD. All experiments were performed in triplicates. *p < 0.05, **p < 0.01.

Article Snippet: To confirm the above bioinformatics results, the human CRC cDNA array from Origene was used to detect the ETV4 mRNA expression, and the results showed that ETV4 was indeed remarkably elevated in CRC samples compared with the normal colorectal tissues (p<0.001, Fig. d), and of note, the expression level of ETV4 mRNA was positively correlated with lymphatic metastasis (p<0.001, Fig. e) and pathologic grades (p<0.001, Fig. f).

Techniques: Quantitative Proteomics, Over Expression, RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Knockdown, Migration, Invasion Assay

Journal: Frontiers in Pharmacology

Article Title: Estrone-3-Sulfate Stimulates the Proliferation of T47D Breast Cancer Cells Stably Transfected With the Sodium-Dependent Organic Anion Transporter SOAT (SLC10A6)

doi: 10.3389/fphar.2018.00941

Figure Lengend Snippet: SOAT mRNA expression in breast cancer. SOAT mRNA expression was analyzed in the TissueScan TM Breast Cancer cDNA Arrays I-IV, including 176 tumor cDNAs with different classifications (histopathology, grade, stage, and receptor status). Expression of SYMPK was used as endogenous control and ΔC T values are depicted at the y -axis. A cut-off was set at C T of 40. Sub-analyses were performed, including (A) tumor grade and stage, (B) receptor status for ER, PR, HER2 and triple negative breast cancer (TN), and (C) age and ethnos. As the cDNA arrays were not equally distributed for the analyzed subgroups, every single value is depicted for better clarity and additional box-whiskers-plots are given. For analysis of statistical significance, one-way ANOVA with Tukey’s multiple comparisons test was performed. Differences with p < 0.05 were not detected.

Article Snippet: In order to analyze SOAT expression in breast cancer, the following TissueScan TM cDNA arrays were commercially obtained from OriGene (Rockville, MD, United States): Breast Cancer cDNA Array I (BCRT101), Breast Cancer cDNA Array II (BCRT102), Breast Cancer cDNA Array III (BCRT103), and Breast Cancer cDNA Array IV (BCRT104).

Techniques: Expressing, Histopathology

Journal: JNCI Journal of the National Cancer Institute

Article Title: Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer

doi: 10.1093/jnci/djs297

Figure Lengend Snippet: EPHA3 expression in human non–small cell lung cancer (NSCLC). A) EPHA3 expression in paired human lung tumors and normal tissues. A panel of cDNAs from 24 paired samples of human lung tumors and normal lung tissue was purchased from Origene Technologies Inc (TissueScan lung cancer tissue qPCR array IV-matched pairs). EPHA3 expression in these patient samples was measured by quantitative polymerase chain reaction (qPCR) and presented as relative expression level normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on a log2 scale. B) EPHA3 expression in different stages of lung cancer. Two panels of cDNAs from 15 normal lung tissue and 70 cancer samples were purchased from Origene Technologies Inc (TissueScan lung cancer tissue qPCR array III and V). EPHA3 expression was measured by quantitative PCR and is presented as the relative expression level normalized to GAPDH. Shown are the data from individual samples (each symbol representing one patient) and the median values stratified according to clinical stage and plotted relative to similar analyses of a tissue collection of normal lung samples Figure 2 (continued).(P < .001, two-sided Kruskal–Wallis test followed by two-sided individual Mann–Whitney tests). C) EPHA3 protein expression in paired tumor and normal lung tissues. EPHA3 expression was analyzed in nine paired lung cancer and nontumor lung tissues by immunohistochemistry using a monoclonal anti-EPHA3 antibody, the specificity of which was verified in Supplementary Figure 3, ​,3B3B (available online). Shown are representative photomicrographs from two out of a total of nine pairs of tumor samples that could be matched with non-tumor lung tissue from the same patient. Arrowheads indicate examples of EPHA3-positive cells (brown) in normal lung tissues. Scale bar on the left two panels: 50 µm; scale bar on the right four panels: 20 µm. D and E) EPHA3 expression in a human lung cancer tissue microarray. EPHA3 expression in tumor samples was assessed by immunohistochemistry in a tissue microarray containing tumor tissue from 104 lung cancer micro-samples and 26 normal lung samples. A representative photomicrograph of EPHA3 immunohistochemistry in tumor or normal tissue is shown in (D). Scale bar: 20 µm. The aggregate numbers of anti-EPHA3 positive (red) and negative (blue) samples in normal and cancer samples are shown in (E) (P = .005, two-sided χ2 test).

Article Snippet: A panel of cDNAs from 24 paired samples of human lung tumors and normal lung tissue was purchased from Origene Technologies Inc (TissueScan lung cancer tissue qPCR array IV-matched pairs).

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunohistochemistry, Microarray