pcdna3.1 plasmids harboring the catalytically inactive trex1 d18n mutant genscript omu22134m  (GenScript corporation)

Journal: PLOS ONE

Article Title: A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses

doi: 10.1371/journal.pone.0305962

Figure Lengend Snippet: A) TREX1 Western blot demonstrating overexpression of both wild-type mouse TREX1 and mouse TREX1 carrying the D18N inactivating mutation. Endogenous mouse TREX1 present in a J774 cellular lysate is shown as reference to confirm the size of the upper band as full-length mouse TREX1. α-tubulin was used as a loading control. B–D) Exonuclease assays performed with HEK293T mouse TREX1 overexpression or empty vector lysates demonstrate the effects of VB-85680 and VB-85662 on B) wild-type mouse TREX1 (5 ng/μL total protein), C) Catalytically inactive mouse TREX1 D18N (100 ng/μL total protein) and, D) endogenous exonuclease activity (100 ng/μL total protein). E) Western blot for endogenous levels of cGAS,STING and TREX1 in both 4T1 and THP1-dual cells. F) Endogenous exonuclease activity in 4T1 cell lysates is inhibited by VB-85680, but not by VB-85662. Compounds were titrated in the presence of 6 μg 4T1 cytosolic lysate using the 3’ to 5’ Exonuclease Activity Assay from BioVision. G) Inhibition of endogenous human TREX1 exonuclease activity by VB-85680 in THP1-Dual™ and THP1-Dual™ TREX1 knockout cell lysates. The exonuclease assay was carried out as described in 1E . Error bars for all experiments represent +/- SD.

Article Snippet: TREX1 overexpression in HEK293T cells (ATCC CRL-3216) was achieved via transient transfection of pcDNA3.1 plasmids harboring either wild-type full-length mouse TREX1 (GenScript OMu22134C) or the catalytically inactive TREX1 D18N mutant (GenScript OMu22134M).

Techniques: Western Blot, Over Expression, Mutagenesis, Control, Plasmid Preparation, Activity Assay, Inhibition, Knock-Out

Journal: PLOS ONE

Article Title: A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses

doi: 10.1371/journal.pone.0305962

Figure Lengend Snippet: A ) Activation of the THP1-Dual™ interferon regulatory factor (IRF) pathway by VACV-70 in combination with VB-85680 was monitored by detection of Lucia luciferase by Quanti-Luc gold. Increased activity was only detected using either cGAMP alone or the combination of TREX1 inhibitor and DNA. B) THP1-Dual™ cells were cultured for 2-days in various serum concentrations followed by transfection with 1200 ng/mL VACV-70 for an additional 18 hours. The Quanti-Luc™ assay was used to demonstrate increased sensitivity of the reporter to exogenous VACV-70 DNA under low serum conditions. C ) A WST1 assay was run in parallel to demonstrate the relative health of cells cultured in lower serum conditions and transfection with VACV-70 DNA. D ) THP-1-Dual™ cells were cultured for 72 hours in either 10% serum or 1% serum. Western blotting was used to evaluate the expression of TREX1 over time in cells grown in each medium. TREX1 protein levels increased in THP1-Dual™ cells cultured under low serum conditions. E) The Quanti-Luc™ assay was used to assess reporter responsiveness to G3-YSD transfection relative to VACV-70. F) THP1-Dual™ cells were cultured for 3 days under low serum conditions. The cells were then batch-transfected with 10 ng/mL G3-YSD and treated with decreasing doses of VB-85680, starting at 30 μM. The IC 50 of VB-85680 was determined to be 2.9 μM. Error bars for all experiments represent +/- SD.

Article Snippet: TREX1 overexpression in HEK293T cells (ATCC CRL-3216) was achieved via transient transfection of pcDNA3.1 plasmids harboring either wild-type full-length mouse TREX1 (GenScript OMu22134C) or the catalytically inactive TREX1 D18N mutant (GenScript OMu22134M).

Techniques: Activation Assay, Luciferase, Activity Assay, Cell Culture, Transfection, Western Blot, Expressing

Journal: PLOS ONE

Article Title: A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses

doi: 10.1371/journal.pone.0305962

Figure Lengend Snippet: Expression of IFIT1 in THP1-Dual™ cells treated with DNA alone or in combination with VB-85680 was measured by Western blot. A) Time-course of IFIT1 expression in THP1-Dual™ cells treated with 1200 ng/mL VACV-70, 10 μM VB-85680, or both. cGAMP (10 μM)-treated THP1-Dual™ cells were used as a positive control. The combination of VB-85680 and VACV-70 increased the expression of IFIT1. B) THP1-Dual™ cells were treated with varying concentrations of VB-85680 in the presence or absence of VACV-70 for 24 hours. Cells were collected and subjected to Western blot analysis of IFIT1 expression. IFIT1 expression was only seen in the presence of TREX1 inhibitor and DNA stimulus. Actin was used as a loading control.

Article Snippet: TREX1 overexpression in HEK293T cells (ATCC CRL-3216) was achieved via transient transfection of pcDNA3.1 plasmids harboring either wild-type full-length mouse TREX1 (GenScript OMu22134C) or the catalytically inactive TREX1 D18N mutant (GenScript OMu22134M).

Techniques: Expressing, Western Blot, Positive Control, Control