pgl2 promoter control vector  (Promega)

Journal: PLoS ONE

Article Title: Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

doi: 10.1371/journal.pone.0044951

Figure Lengend Snippet: A , Expression of CDO1 in CRC cell lines was examined by RT-PCR analysis. No basal expression of CDO1 was seen in all CRC cell lines, and all these cell lines harbored CDO1 methylation (M). Silenced CDO1 was reactivated after treatment with the demethylating agent, 5-Aza-dC, indicating that CDO1 methylation correlates tightly with loss of gene expression in CRC cell lines. β-actin was used as a loading control. m, cells treated with vehicle only; a, cells treated with 5-Aza-dC (5 µM for three days). L, 1 Kb Plus DNA ladder. B , Methylation status of individual CpGs of the island in 5 CRC cell lines and 21 pairs of primary CRC (PT) and their corresponding normal colon tissues (PN) is shown. A total of 38 CGs were numbered from the first to last CG in the sequences as indicated. Black circle, methylation; white circle, unmethylation; grey circle, co-existence of methylated and unmethylated alleles; dashed circle, undetermined. C , Analysis of CDO1 promoter activity by luciferase reporter assay in CDO1 -negative (HCT116) and -positive (HEK293) cells. The promoter constructs (pGL2- CDO1 -#1 and -#2) were pre-treated with or without Sss I methylase for 8 hrs before transfection. High activity of the CDO1 promoter was detected in HEK293 where CDO1 was expressed. Data are expressed as fold increase over pGL2-basic activity. Experiments were done in triplicate, and values indicate means ± SD. Mean values are presented. D. Scatter plot of CDO1 methylation levels in tissues and cell lines (CL) (left). TaqMan methylation values (TaqMeth V) is described in Materials and Method. TaqMan-MSP was performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results. PT, primary CRC; PN, matched normal colon tissues from colon cancer patients; NN, normal colon epithelium from non-cancer patients. Line indicates the optimal cut-off value for CDO1 calculated from ROC analysis. Sample numbers showing TaqMeth V over the cut-off are indicated. The overall TaqMeth V detected in PT was significantly higher than that in PN (right). TaqMan value of two NN (22%, 2/9) was above the cut-off value (24.56 and 17.86 each), suggesting that a low level of CDO1 methylation can be caused by other unknown mechanisms. E , ROC curve analysis of TaqMeth V of CDO1 in CRC. The Area under the ROC (AUROC) conveys the accuracy in distinguishing matched normal colon (PN) from CRC (PT) in terms of its sensitivity and specificity ( P<0.001 ). Solid line, CDO1 ; dashed line, no discrimination. F , Methylation levels of normal (PN) and tumor tissues (PT) in individual patients.

Article Snippet: The pGL2 promoter control vector (Promega, Madison, WI) was digested with both KpnI and XhoI and treated with calf intestinal alkaline phosphatase.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, Activity Assay, Luciferase, Reporter Assay, Construct, Transfection

Journal: iScience

Article Title: Differential regulation of alternate promoter regions in Sox17 during endodermal and vascular endothelial development

doi: 10.1016/j.isci.2022.104905

Figure Lengend Snippet: Sox17 CR1 and CR2 exhibit promoter activity in vitro (A) The 8-day mESC differentiation protocol used to derive definitive endoderm-like and hematopoietic vascular endothelial-like cells. Terminal cellular identity was confirmed by cellular morphology and lineage-specific markers as indicated (see Figure S6 ). (B) Luciferase-activity readout at days 6–8 of endodermal and endothelial differentiation. CR1 and CR2 DNA sequences were inserted in sense (CR1f, CR2f) or antisense (CR1r, CR2r) orientation. pGL4 backbone vector without a promoter, negative control. pGL2.pro vector with an SV40 promoter, positive control. Endoderm protocol, n = 6. Endothelial protocol, n = 4. (C) Luciferase activity from CR1f and CR2f from three time points during the endodermal differentiation protocol. n = 3. Data are normalized to pGL2.pro. (D) Five conserved motifs within CR2 identified with UCSC conservation track. Six 9-basepair blocks (magenta on wild-type CR2 sequence) were mutated by base transversion (G–A and C–T, or vice versa). (E) Luciferase activity readout at days 6–8 of the endodermal or endothelial differentiation protocol for WT and six block-mutations within the CR2 sequence (m1–m6). pGL4, negative control. pGL4.CR2f, positive control. Endoderm protocol, n = 7. Endothelial protocol, n = 6. (B, C, E) Dot color represents samples differentiated from the same date (same color) or different dates (different colors). Each dot represents one well of differentiated cells. Bars and error bars represent the mean ± SEM. Results were analyzed by a one-way ANOVA. ∗ p < 0.05; ∗∗ p < 0.01. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: pGL2-Control vector , Promega , Cat# E1611.

Techniques: Activity Assay, In Vitro, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Sequencing, Blocking Assay

Journal: iScience

Article Title: Differential regulation of alternate promoter regions in Sox17 during endodermal and vascular endothelial development

doi: 10.1016/j.isci.2022.104905

Figure Lengend Snippet:

Article Snippet: pGL2-Control vector , Promega , Cat# E1611.

Techniques: Immunofluorescence, Staining, Western Blot, Flow Cytometry, Cytometry, Recombinant, Protease Inhibitor, Plasmid Preparation, Purification, Transfection, Reporter Assay, Bicinchoninic Acid Protein Assay, Derivative Assay, Mutagenesis, Clone Assay, Software

Journal: Biology of Reproduction

Article Title: Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells †

doi: 10.1093/biolre/ioy097

Figure Lengend Snippet: DNA end-joining efficiency is decreased in DES vs. VEH MSCs. Luciferase gene reporter assay detected lesser DNA end-joining activity in DES vs. VEH MSCs following co-transfection with linearized HindIIIpGL2-control luc and CMV-β-gal plasmids. Means of combined experimental and biological replicates were compared using a two-tailed Student unpaired t-test. Lines represent the mean normalized end-joining activity ± SD. *P < 0.0001

Article Snippet: Plasmid DNA preparation Briefly, SV40- luc pGL2-control vector plasmid DNA ( Supplemental Figure S1A , Promega, Madison, WI) was used to transform DH5α Escherichia coli cells for plasmid amplification and was then isolated using PureYield Plasmid Midiprep System (Promega).

Techniques: Luciferase, Reporter Assay, Activity Assay, Cotransfection, Two Tailed Test