Journal: iScience

Article Title: TRAF7 is an essential regulator of blood vessel integrity during mouse embryonic and neonatal development

doi: 10.1016/j.isci.2023.107474

Figure Lengend Snippet:

Article Snippet: COS-1 African green monkey kidney cell line , Sigma-Aldrich , Cat#88031701.

Techniques: Produced, Affinity Purification, Recombinant, Transfection, Mutagenesis, Expressing, esiRNA, TaqMan Assay, Real-time Polymerase Chain Reaction, Knock-Out, Plasmid Preparation, Software

Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: Mechanism of replication initiated from the HPV18 ori depends on the size of the replicon. (A) Transient replication of the HPV18 ori harboring replicons with different sizes in the U2OS cells. pUCURR18 (4kb), pGLURR18 (6kb), or pGEXURR18 (8,2 kb) were transfected together with the HPV18 E1 and E2 expression vectors into the U2OS cells. Episomal DNA was extracted 72h after the transfection, digested with replicon non-cutter (left panel) or single cutter enzyme (right panel), combined with DpnI to digest the non-replicated plasmids, resolved in 0.8% agarose gel and analyzed by SB. Asterisks represent migration of the monomeric closed circular forms of the respective replicons. (B) Neutral-neutral 2D analysis of the replication intermediates arising from the HPV18 harboring replicons of different sizes during the transient replication in the U2OS cells and the SV40 origin containing pCDNA3 replicon in Cos1 cells. The replication intermediate structure that varies between different HPV replicons is indicated with an arrow.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Transfection, Expressing, Agarose Gel Electrophoresis, Migration

Journal: Toxicology in vitro : an international journal published in association with BIBRA

Article Title: Development of a novel recombinant cell line for detection and characterization of Ah receptor nuclear translocation in intact cells

doi: 10.1016/j.tiv.2020.104873

Figure Lengend Snippet: The functional analysis of YFP-tagged AhR. (A) Confirmation of the expression of in vitro expressed AhRy and yAhR proteins. Wild-type AhR (wtAhR) and C-term or N-term YFP-tagged AhR (AhRy and yAhR, respectively) were synthesized in vitro with 35S-methionine and an aliquot each protein lysate (1 μl) was analyzed by SDS-PAGE, and 35S-labeled proteins visualized by Phosphoimager analysis. The results shown are representative of three independent experiments. (B) Confirmation of DNA binding of AhR complexes. wtAhR, AhRy, yAhR and mouse wtArnt proteins were expressed in vitro, and each AhR were mixed with Arnt (1:1) and incubated with DMSO (2% (v/v)) or TCDD (20 nM) for 3 h at room temperature. TCDD-inducible protein-DNA complex formation (AhR:Arnt:DRE) were resolved by gel retardation assay as described in Material and Methods. (C) Cos-1 cells were transiently transfected with wtAhR, AhRy or yAhR expression plasmid, the AhR-responsive firefly luciferase reporter plasmid pGudLuc6.1 and Renilla luciferase reporter plasmid pRL-TK (for transfection normalization). After 24 h, transfected cells were incubated in the presence of DMSO (0.1% (v/v) or TCDD (1nM) for 24 h followed by analysis of luciferase activity. Firefly luciferase activity was divided by that of Renilla luciferase and the resulting values expressed as the mean ± SD of three replicate transfections.

Article Snippet: The AhR deficient monkey kidney cell line (Cos-1) (American Type Culture Collection) and the AhR defective mouse hepatoma cell line, TAOc1BP r c1 (TAO) [ Miller et al., 1983 ] were grown and maintained in α-Minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Functional Assay, Expressing, In Vitro, Synthesized, SDS Page, Labeling, Binding Assay, Incubation, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Biochemical and biophysical research communications

Article Title: A direct comparison of the transcriptional activities of progestins used in contraception and menopausal hormone therapy via the mineralocorticoid receptor

doi: 10.1016/j.bbrc.2020.03.100

Figure Lengend Snippet: (A) Like P4, all the selected progestins compete with [3H]-Ald for binding to the human MR. COS-1 cells were transiently transfected with 11.25 μg of the human MR expression vector and incubated for 16 hours with 0.2 nM [3H]-Ald in the absence or presence of increasing concentrations of unlabelled ligands. Counts per minute (cpm) were normalized to the protein concentration (mg/ml). Total specific binding of [3H]-Ald only was expressed as 100% and the binding of unlabelled competitors expressed as a percentage relative to this. (B) Log Kd/Ki values of the ligands for the MR are plotted.

Article Snippet: The COS-1 monkey kidney cell line was obtained from the ATCC and was previously described [ 6 ].

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Protein Concentration

Journal: Biochemical and biophysical research communications

Article Title: A direct comparison of the transcriptional activities of progestins used in contraception and menopausal hormone therapy via the mineralocorticoid receptor

doi: 10.1016/j.bbrc.2020.03.100

Figure Lengend Snippet: Most progestins display differential antagonist potencies for transactivation via the MR, while DRSP and P4 are weak partial MR agonists for transactivation. COS-1 cells, transiently transfected with 1 μg of the human MR expression vector and 10 μg of the pTAT-2xPRE-E1b-luciferase reporter plasmid, were treated with increasing concentrations of Ald, P4 or progestins in the absence (A) or (C) presence of 1 nM Ald (set as 100%) for 24 hours. (B) Induction by P4 and the progestins at 10 μM (from A) is shown as a percentage relative to 10 μM Ald expressed as 100%. Luciferase activity was measured in relative light units and normalized to the protein concentration. (D) Maximal responses and (E) log EC50 values of the ligands for the MR (from C) were plotted.

Article Snippet: The COS-1 monkey kidney cell line was obtained from the ATCC and was previously described [ 6 ].

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Protein Concentration

Journal: Biochemical and biophysical research communications

Article Title: A direct comparison of the transcriptional activities of progestins used in contraception and menopausal hormone therapy via the mineralocorticoid receptor

doi: 10.1016/j.bbrc.2020.03.100

Figure Lengend Snippet: Most progestins display indistinguishable agonist activity for transrepression via the MR on the synthetic NFκB, but not the AP-1, promoter. COS-1 cells transiently transfected with 1.35 μg of the human MR expression vector and 2.7 μg of the (A) 7xAP-1-luciferase or (C) 5xNFκB-luciferase reporter plasmid, were treated with EtOH and 10 ng/ml PMA in the absence (set as 100%) or presence of increasing concentrations of ligands for 24 hours. Luciferase activity was measured and normalized as in Fig. 2. Treatment with 10 ng/ml PMA resulted in a ~14-fold and ~32-fold induction, respectively (Fig. 3A and 3C inserts). (B and D) Log EC50 values of the ligands for the MR were plotted.

Article Snippet: The COS-1 monkey kidney cell line was obtained from the ATCC and was previously described [ 6 ].

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