human colon cancer cdna array  (OriGene)

Journal: International Journal of Molecular Sciences

Article Title: The Study of Chromobox Protein Homolog 4 in 3D Organoid Models of Colon Cancer as a Potential Predictive Marker

doi: 10.3390/ijms26157385

Figure Lengend Snippet: CBX family genes expression in colon cancer. ( A ) CBX expression level by qRT-PCR in three colon cancer cell lines (CACO-2, SW480, and HCT 116) compared to a normal colon cell line (NMC 460D). ( B ) CBX gene expression level in a colon cancer cDNA RT-qPCR array consisting of tumor ( N = 40) and normal samples ( N = 8). ( C ) Expression analysis of CBX family genes of colon tumor in UALCAN dataset (Normal = 41, Primary Tumor = 286). The star symbol indicates statistical significance (**: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

Article Snippet: Human TissueScan Colon Cancer Tissue qPCR Panel IV (HCRT304), containing first-strand cDNA from 48 samples covering 8-normal, 5-Stage I, 8-IIA, 1-II, 1-IIIA, 6-IIIB, 3-IIIC, 6-III, and 10-IV patients, was purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Study of Chromobox Protein Homolog 4 in 3D Organoid Models of Colon Cancer as a Potential Predictive Marker

doi: 10.3390/ijms26157385

Figure Lengend Snippet: Association between CBX4 expression levels and clinical stage and grade of CRC patients. ( A ) CBX4 expression analyses performed using the UALCAN dataset with relative statistical comparison among groups. ( B ) CBX4 expression analyses in a colon cancer cDNA RT-qPCR array consisting of tumor ( n = 40) and normal samples ( n = 8) stratifying patients according to stage and grade. The star symbol indicates statistical significance (**: p ≤ 0.01).

Article Snippet: Human TissueScan Colon Cancer Tissue qPCR Panel IV (HCRT304), containing first-strand cDNA from 48 samples covering 8-normal, 5-Stage I, 8-IIA, 1-II, 1-IIIA, 6-IIIB, 3-IIIC, 6-III, and 10-IV patients, was purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Comparison, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: The Study of Chromobox Protein Homolog 4 in 3D Organoid Models of Colon Cancer as a Potential Predictive Marker

doi: 10.3390/ijms26157385

Figure Lengend Snippet: Characterization of CBX4 in CRC organoids. ( A ) Representative images of ex vivo PDO culture obtained from colorectal cancer biopsies are reported. Scale bar: 10 µm. ( B ) Real-time qPCR analysis of CBX4 in two groups of PDOs: the first one derived from healthy tissues (CNT) and the second one derived from tumor tissues (Tumor). Results were normalized to RPS18 mRNA and analyzed by 2 −ΔΔCt method. ( C ) Analysis of the knockdown efficiency of siCBX4 assessed by RT-PCR in PDOs after 72 h. ( D ) Effect of CBX4 silencing on PDO viability assessed by ATP Lite assay after 72 h. The star symbol indicates statistical significance (**: p ≤ 0.01).

Article Snippet: Human TissueScan Colon Cancer Tissue qPCR Panel IV (HCRT304), containing first-strand cDNA from 48 samples covering 8-normal, 5-Stage I, 8-IIA, 1-II, 1-IIIA, 6-IIIB, 3-IIIC, 6-III, and 10-IV patients, was purchased from Origene (Rockville, MD, USA).

Techniques: Ex Vivo, Derivative Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: The Study of Chromobox Protein Homolog 4 in 3D Organoid Models of Colon Cancer as a Potential Predictive Marker

doi: 10.3390/ijms26157385

Figure Lengend Snippet: Role of CBX4 silencing in CRC organoids ( A , B ). Representative images of our target molecules through immunofluorescence analysis. Localization of NFkB in tumor ex vivo PDOs before (CNT) and after siCBX4. Maximal projection images of PDO incubated NFkB (green signal) and cell nuclei were stained with Hoechst 33342 (blue signal). Scale bar: 10 µm. ( C ) Real time qPCR analysis of NF-κB, c-myc, TNF-α, and IL-1 in tumor ex vivo PDOs before (CTR) and after CBX4 silencing. Results were normalized to RPS18 mRNA and analyzed by 2 −ΔΔCt method. The star symbol indicates statistical significance (*: p ≤ 0.05, **: p ≤ 0.01).

Article Snippet: Human TissueScan Colon Cancer Tissue qPCR Panel IV (HCRT304), containing first-strand cDNA from 48 samples covering 8-normal, 5-Stage I, 8-IIA, 1-II, 1-IIIA, 6-IIIB, 3-IIIC, 6-III, and 10-IV patients, was purchased from Origene (Rockville, MD, USA).

Techniques: Immunofluorescence, Ex Vivo, Incubation, Staining

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: BMC cancer

Article Title: Nitric-Oxide Synthase trafficking inducer (NOSTRIN) is an emerging negative regulator of colon cancer progression.

doi: 10.1186/s12885-022-09670-6

Figure Lengend Snippet: Fig. 2 Real time PCR array analysis for profiling Epithelial-Mesenchymal transition (EMT) signature genes in NOSTRIN over-expressed colon cancer cell line. A Quantitative real time PCR analysis of Nostrin using RNA from empty vector transfected (control) and Nostrin cDNA transfected HCT116 cells. GAPDH was used as an endogenous control for normalization. B Western blot analysis of NOSTRIN using cell lysates from empty vector transfected (control) and Nostrin cDNA transfected HCT116 cells. GAPDH was used as an endogenous control. Full length blots are shown in Fig. S3. Quantification of the protein level from the western blot was done using NIH ImageJ software and is represented using bar graphs adjacent to the blot; Error bars represent standard error of mean from three biological replicates. **p < 0.01, ***p < 0.001. C Scatter plot of real time PCR array using control and Nostrin overexpressing HCT116 cells showing differential EMT gene expression pattern. Genes with similar expression patterns are represented by black dots that are close to the line of regression while yellow and blue dots are for the up-regulated and down-regulated genes, respectively. D Functional annotation of 16 genes that are differentially regulated in all three experiments upon over-expression of NOSTRIN in HCT116 cells

Article Snippet: Colon cancer progression through various stages is associated with reduced NOSTRIN mark To corroborate our ex vivo findings with in vivo colon cancer progression, we performed a real time PCR based human colon cancer cDNA array (OriGene, USA) containing cDNAs from different stages of colon cancer patients including those from normal colon samples, comparing changes in Nostrin transcripts among those patient samples.

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Control, Western Blot, Software, Gene Expression, Expressing, Functional Assay, Over Expression