ABSTRACT: To evaluate whether FEAT has cellular functions relevant in oncogenesis, FEAT was overexpressed in NIH3T3 cells, which only weakly express FEAT protein, and the alterations in genome-wide transcriptional profiles were analyzed by microarrays. Overall design: The ORF of human FEAT cDNA was subcloned into the pENTR3C entry vector of the Gateway cloning system (Invitrogen). LR recombination with the pEF-DEST51 vector (Invitrogen) yielded the pEF-DEST51-FEAT plasmid that encodes FEAT driven by the elongation factor 1alpha promoter. NIH3T3 cells were transfected with pEF-DEST51-FEAT using HilyMax (Dojindo Laboratories). After 7 days of selection with 10 µg/ml blasticidin S (Kaken Pharmaceutical), colonies were picked and screened for clones in which FEAT protein was overexpressed. Three clones, FEAT-3, -15, and -20 were used in further studies. The ccdB gene in the pEF-DEST51 vector was deleted to construct the pEF-DEST51-∆ccdB plasmid, which was stably transfected into NIH3T3 cells to obtain control cell lines, named ∆ccdB-1, -2, and -3.
Project description:Transgelin was the top-ranked marker of metastatic potential identified in the comparison of node-positive colorectal cancer (CRC) versus node-negative CRC in our previous study. Transgelin is localized in the nucleus of cultured CRC cells and microRNA-mediated knockdown of TAGLN (the gene encoding transgelin) expression modulates the expression of genes involved in the epithelial-to-mesenchymal transition. We performed gene expression profiling on control and transgelin-overexpressing RKO cells using Affymetrix microarray technology. The plasmid carrying TAGLN was generated using the pcDNA6.2/EmGFP-Bsd/V5-DEST vector (Invitrogen, Carlsbad, CA) and the pDONR221-TAGLN-mut plasmid from a previous study by an LR recombination reaction (http://www.invitrogen.com). After lipofectamine-mediated transfection of the TAGLN-carrying plasmid or the vector-only control plasmid, stable transfectants were selected and cultured in medium containing 25 μg/ml blasticidin followed by RNA extraction and hybridization on Affymetrix microarrays. Six samples were obtained from three independent experiments (technical replicates).
Project description:The yeast expression vectors used to express AAE13 was constructed as follows: AAE13 (At3g16170) was amplified from the vector pENTR-AAE13. The amplified product was cloned into pCR®8⁄GW⁄TOPO® (Invitrogen). The resulting plasmid was combined with the Advanced Gateway destination vectors pAG305GPD-ccdB (Addgene, Boston, MA) in an attL X attR recombination reaction to generate the vectors pAG305GPD-AAE13. The integrating vector pAG305GPD-AAE13 was introduced into WAT11, which was also transformed empty vector pAG305GPD-ccdB as control (B1, B2, B3 and B4). The transgenic and control yeast cells were cultured in SD-drop out medium at 30 °C under shaken conditions, malonate was fed into cells at final concentration 0.2 mM when the value of OD600 reached at 0.1. The cells were harvested at mid-log phase. Two-condition experiment, Transgenic yeast (pAG305GPD-AAE13) vs. control yeast (pAG305GPD-ccdB). Biological replicates: 4 control replicates, 4 transgenic replicates.
Project description:To investigate the effect of STAT3 activation on the expression of gastric cancer cells, expression profile was compared in MKN28 cells overexpressed with control vector vs mouse constitutively activated STAT3 mutant (STAT3c). MKN28 gastric cancer cells were transfected with pcDNA3.1 (vector control) or plasmid overexpressing STAT3c (treatment). Stable clones were selected for RNA extraction and expression microarray analysis (Agilent). Experiments were repeated twice.
Project description:The aim was to identify gene expression changes upon expression of KIAA1199 in a colon cancer cell line. Inducible expression of KIAA1199A was achieved with a two-step procedure. First, SW480 cells were transfected with the pcDNA6TR vector (Invitrogen) and the tetracycline repressor-expressing cells selected with 7 μg/ml blasticidin S (InvivoGen). The blasticidin-resistant cells were then transfected with the pT-Rex-DEST30-KIAA1199 construct (see below) and selected with 0.4 mg/ml G418. The growth medium used for these cells was supplemented with 10% Tet-system-approved fetal calf serum (Biochrom).
Project description:We aimed to analyze the relationship between TET1 and aberrant CpG methylation in colorectal cancer (CRC). We established two stable TET1 knockdown clones and negative control clones of HCT116 cells, and carried out DNA methylation analysis with HumanMethylation450 BeadChip. Overall design: RNAi-induced TET1 knockdown was accomplished using a BLOCK-iT Pol II miR RNAi Expression Vector kit (Thermo Fisher Scientific). Two sets of oligonucleotides targeting TET1 were purchased from Invitrogen and ligated into a pcDNA6.2-GW/EmGFP-miR vector (Thermo Fisher Scientific). Cells were transfected with each TET1 knockdown vector or a pcDNA6.2-GW/EmGFP-miR-neg control plasmid (Thermo Fisher Scientific), after which they were selected with 0.6 mg/ml G418. GFP-positive colonies were isolated, and knockdown efficiencies were analyzed using RT-qPCR.
Project description:To determine genes regulated by HNF4G, we retrovirally infected LNCaP cells in triplicates to express HNF4G and empty vector as control. cDNA for HNF4G in pDONR201 vector was obtained from Harvard medical school Plasmid database (ID:HsCD00022314) and was cloned into an murine stem cell virus (MSCV)-based retroviral vector with puromycin selection marker (Addgene) using Gateway technology. Overall design: LNCaP cells were infected with retroviruses in triplicates for HNF4G and control empty vector expression. After three days of infection cells were selcted with puromycin for two days and later on RNA was harvested for gene expression analysis.
Project description:HEK293 cells were transfected with control plasmid (pcDNA1/Neo; Invitrogen) or with the plasmid encoding HCaRG by a standard calcium phosphate co-precipitation method. The clones expressing the highest levels of HCaRG, HCaRG clone 8 and 9 were used in this experiment, while clone transfected with vector alone, Neo clone, served as controls. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen).
Project description:Global DNA hypomethylation and DNA hypermethylation of promoter regions—including tumor suppressor genes—are frequently detected in human cancers. Although many studies have suggested a contribution to carcinogenesis, it is still unclear whether the aberrant DNA hypomethylation observed in tumors is a consequence or a cause of cancer. We found that overexpression of Stella (also known as PGC7, Dppa3), a maternal factor required for the maintenance of DNA methylation in early embryos, induced global DNA hypomethylation and transformation in NIH3T3 cells. This hypomethylation was due to the binding of Stella to Np95 (also known as Uhrf1, ICBP90) and the subsequent impairment of Dnmt1 localization. In addition, enforced expression of Stella enhanced the metastatic ability of B16 melanoma cells through the induction of metastasis-related genes by inducing DNA hypomethylation of their promoter regions. Such DNA hypomethylation itself causes cellular transformation and metastatic ability. These data provide new insight into the function of global DNA hypomethylation in carcinogenesis. We used microarrays to detail the global programme of gene expression by PGC7/Stella overexpression. RNA was extracted from NIH3T3 or B16F10 murine cell lines overexpressed PGC7/Stella and was hybridized on Affymetrix microarrays. We compared gene expression levels between control and PGC7/Stella-overexpressed cells. Microarray analysis was performed in NIH3T3 cells including two independent Stella-expressing NIH3T3 clones and a mixture of Stella-expressing NIH3T3 clones and in B16-F10 cells including three independent Stella-expressing B16-F10 clones.
Project description:We aimed to analyze the relationship between TET1 and aberrant CpG methylation in colorectal cancer (CRC). We established two stable TET1 knockdown clones and negative control clones of HCT116 cells, and carried out gene expression analysis with Agilent Human Gene Expression microarray kit. Overall design: RNAi-induced TET1 knockdown was accomplished using a BLOCK-iT Pol II miR RNAi Expression Vector kit (Thermo Fisher Scientific). Two sets of oligonucleotides targeting TET1 were purchased from Invitrogen and ligated into a pcDNA6.2-GW/EmGFP-miR vector (Thermo Fisher Scientific). Cells were transfected with each TET1 knockdown vector or a pcDNA6.2-GW/EmGFP-miR-neg control plasmid (Thermo Fisher Scientific), after which they were selected with 1.0 mg/ml G418. GFP-positive colonies were isolated, and knockdown efficiencies were analyzed using RT-qPCR.