Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: Sequencing results of the inserts in CD147-TT-pSICHECK2 plasmid. Arrows indicate the inserted rs6757 TT genotype fragment

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Sequencing, Plasmid Preparation

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The Blast of the inserts in CD147-TT-pSICHECK2 plasmid. The sequencing results were analyzed by Blast, the inserted sequence of target gene was 100% consistent with the sequence of CD147 rs6757 TT genotype in the NCBI database

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Plasmid Preparation, Sequencing

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: Sequencing results of the inserts in CD147-CC-pSICHECK2 plasmid. The sequencing result showed that the mutation from nucleotide T to C at the target site (marked in green) of CD147 gene had successfully achieved

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Sequencing, Plasmid Preparation, Mutagenesis

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The Blast of the inserts in CD147-CC-pSICHECK2 plasmid. After mutating from nucleotide T to C, the inserted sequence was 99% consistent with the sequence of CD147 in the NCBI database by Blast analysis

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Plasmid Preparation, Sequencing

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The R/F analysis of a luciferase reporter vector. A Luciferase expression was significantly reduced following transfection with CD147-TT-pSICHECK2 ( P < 0.01); the translational suppression of miR-3976 can be reversed by its inhibitor. B There was no significant statistical difference between the groups following transfection with CD147-CC-pSICHECK2. C Luciferase activity was decreased by miR-3976 in dose-dependent manner for the constructs with a TT genotype but not changed for constructs with a CC genotype at the rs6757:T>C polymorphism. Each transfection was carried out in triplicate. * denotes a p value < 0.05; ** denotes a p value < 0.01

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Luciferase, Plasmid Preparation, Expressing, Transfection, Activity Assay, Construct

Journal:

Article Title: Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence

doi:

Figure Lengend Snippet: IA-1 zinc-fingers 2 and 3 are essential for transcriptional activity. Various zinc-finger constructs were fused in frame with hEgr-1 AD (a.a. 1–147). Co-transfection of zinc-finger mutants with the IA-1 –426/+40 bp promoter/pCAT3 reporter gene into β-TC-1 cells revealed that zinc-fingers 2 and 3 are the key motifs that contribute to the transcriptional activity. Zinc-finger 3 alone exhibits 50% of the control activity, whereas the combination of zinc-finger 3 and 4 only exhibits 25% of the control activity. The data are expressed as fold increase over the empty pcDNA3 expression vector. A CMV–βgal vector is used to normalize transfection efficiency. The graph represents the average of three separate experiments and SEM.

Article Snippet: The –426/+40 bp IA-1 promoter/CAT construct was generated by subcloning a Nhe I/ Xho I –426/+40 bp IA-1 promoter fragment into the Nhe I/ Xho I site of the pCAT3 vector (Promega).

Techniques: Zinc-Fingers, Activity Assay, Construct, Cotransfection, Expressing, Plasmid Preparation, Transfection

Journal: Oncotarget

Article Title: microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck cancer

doi: 10.18632/oncotarget.6972

Figure Lengend Snippet: A. Luciferase assays were performed with pmirGLO vector containing the 3′-UTR of NUAK1 and the graph shows the relative luciferase activity in mature miR-203- or control miRNA-transfected cells. The data were normalized to control samples and results are presented as means ± SD. ** P < 0.01. B. NUAK1 expression was examined in control- or mature miR-203-transfected HNSCC cells (KOSCC25B, SpSCC, and HOC313) by western blotting analysis. The graph shows the NUAK1/β-actin ratio by densitometric analysis. C. NUAK1 expression was examined in control- or miR-203 inhibitor-transfected HSC2 cells by western blotting analysis. The graph shows the NUAK1/β-actin ratio by densitometric analysis.

Article Snippet: The oligonucleotides had the following sequences: SNAI2-1: 5′-CATTGCTGCCAAATCATTTCAA-3′ SNAI2-2: 5′-TTACTATTTTAAAACATTTTAA-3′ SNAI2-3: 5′-TAATGTACTTAAACTATTTCAA-3′ The full-length 3′-UTR of human NUAK1 was amplified by PCR ( ) from genomic DNA and cloned at the EcoRI site into the pmirGLO vector (Promega).

Techniques: Luciferase, Plasmid Preparation, Activity Assay, Transfection, Expressing, Western Blot

Journal: Gene Expression

Article Title: An In Vitro Approach to Test the Possible Role of Candidate Factors in the Transcriptional Regulation of the RET Proto-Oncogene

doi:

Figure Lengend Snippet: RET minimal promoter and interaction with nuclear extracts from SK-N-MC cells. (A) The sequence of the basal RET promoter has been divided into seven partially overlapping oligonucleotides numbered from 0 to 6. Arrows point to the starting nucleotide of the relative pGL3basic-RET PROMOTER deleted constructs used in cotransfection experiments with HOX11L1; the canonical SP1 recognition sites are written in italicized capital letters; +1 points to the transcription start site. (B) EMSA performed on a nondenaturing 5% polyacrylamide gel using seven γ-32P-labeled probes overlapping the entire sequence under analysis. (−) nuclear extract from SK-N-MC cells transfected with the empty vector; (+) nuclear extracts from pcDNA3.1TOPO-HOX11L1[V5-His] transfected SK-N-MC cells. (C) Supershift experiments with probe 3 and different antibodies: after incubation with SP1-specific antibody, a supershift is evident (white star) with nuclear extracts from both empty vector transfected (left panel) and HOX11L1 transfected (right panel) cells; in the left panel a typical retarded band for SP3 is shown (black star); each complex is specific because it disappeared after competition with a molar excess of cold probe. In nuclear extracts from pcDNA3.1TOPO-HOX11L1[V5-His] transfected cells, the incubation with the anti-V5 antibody did not produce any modification in the preformed complexes. (D) Cotransfection of the SP1 expression plasmid with pGL3basic-RET PROMOTER(5.1Kb) in SK-N-MC shows a mild increase in Luciferase activity, compared to the value obtained from the cotransfection with the empty vector (3.1 TOPO).

Article Snippet: Construction of Human c- RET Promoter Reporter Plasmids A fragment of the human c- RET 5′ flanking region, previously cloned in p-Bluescript SK − (Stratagene) ( 25 ) (GenBank accession number AF03214), was used to subclone the sequence from −5078 to +53 with respect to RET transcription start site, in the Kpn I- Hin dIII restriction sites of the pGL3basic vector (Promega), upstream of the Firefly Luciferase reporter gene.

Techniques: Sequencing, Construct, Cotransfection, Labeling, Transfection, Plasmid Preparation, Incubation, Modification, Expressing, Luciferase, Activity Assay