guide rna lenticrispr v2 constructs  (GenScript corporation)

Journal: Antioxidants

Article Title: Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

doi: 10.3390/antiox10020334

Figure Lengend Snippet: STAT1 and STAT3 activation in senescent preadipocytes and gene expression validation of their targets in senescent 3T3-L1, stromal vascular fraction (SVF) and human adipose fat tissues. ( A ) Western blot analysis of phospho-STAT1 (Y701), STAT1, phospho-STAT3 (Y705), STAT3 in proliferating and H 2 O 2 treated preadipocytes at different time points. Actin was used as a loading control. ( B ) Gene expression analysis of the most significantly upregulated STAT1/3 targets in senescent preadipocytes by real-time qPCR. Results (fold change vs. control) are presented as means ± SEM from three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (Student’s t-test). ( C ) Gene expression analysis of STAT1/3 targets in SVF of high-fat diet mice by real-time qPCR. Gene expression results (fold change) are presented as means ± SEM from three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (Student’s t-test). ( D ) Table of patient cohort measures selected for the study. Body Mass Index (BMI), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), Mean Arterial Pressure (MAP), Low Density Lipoprotein (LDL), High-Density Lipoprotein (HDL). ( E ) Correlation analysis of STAT1/3 targets gene expression data in human adipose tissue.

Article Snippet: Guide RNA lentiCRISPR v2 constructs targeting STAT1, STAT3, cGAS, STING and Non-targeting (NT) control were purchased from Genscript (Piscataway, NJ, USA) and generated as described previously [ ].

Techniques: Activation Assay, Gene Expression, Biomarker Discovery, Western Blot, Control

Journal: Antioxidants

Article Title: Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

doi: 10.3390/antiox10020334

Figure Lengend Snippet: Antagonistic functions of STAT1 and STAT3 in regulating growth arrest and cell survival phenotypes in senescent preadipocytes. ( A ) Western blot assessing the protein levels of STAT1 and STAT3 in control NT, STAT1 KO, STAT3 KO cells. Actin was used as loading control. ( B ) Growth curve analysis of untreated non-targeting (NT), STAT1 KO, and STAT3 KO preadipocytes. The number of live preadipocytes in each condition was counted on different days and represented as a fold-increase over the number of seeded preadipocytes on Day-1. Results are presented as means ± SEM of three independent experiments. ( C ) Senescence-associated β-galactosidase (SA-β-gal) staining of proliferating (untreated) or H 2 O 2 treated non-targeting (NT), STAT1 KO, and STAT3 KO preadipocytes. SA-β-gal positive preadipocytes were quantified from four different fields and represented as a percentage as indicated within the images. Scale bar: 1000 μm. ( D ) Graph represents the count of cell number of NT, STAT1 KO, and STAT3 KO following H 2 O 2 treatment protocol. Results are presented as means ±SEM of three independent experiments. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001 (ANOVA with post-hoc Tukey test). ( E ) Western blot of phospho-STAT1 (Y701), STAT1, phospho-STAT3 (Y705), STAT3, acetylated p53 (Lys375), total p53, p21 in untreated NT, STAT1 KO, and STAT3 KO preadipocytes vs. H 2 O 2 treated counterparts as indicated. Actin was used as loading control. Results are represented as means ± SEM from three independent experiments.

Article Snippet: Guide RNA lentiCRISPR v2 constructs targeting STAT1, STAT3, cGAS, STING and Non-targeting (NT) control were purchased from Genscript (Piscataway, NJ, USA) and generated as described previously [ ].

Techniques: Western Blot, Control, Staining

Journal: Antioxidants

Article Title: Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

doi: 10.3390/antiox10020334

Figure Lengend Snippet: STAT1 functionally interacts with cGAS/STING to drive the expression of CXCL10 and antiviral response genes and STAT3 negatively regulates this interaction. ( A ) Western blot assessing the protein levels of cGAS, STING, IFNα, IFNβ, CXCL10 and IL6 in untreated NT, STAT1 KO, and STAT3 KO preadipocytes vs. H 2 O 2 treated counterparts as indicated. Actin was used as loading control. Results are represented as means ± SEM from three independent experiments. ( B ) Gene expression analysis of most significantly upregulated SASP molecules in untreated NT, STAT1 KO, and STAT3 KO preadipocytes vs. H 2 O 2 treated counterparts as indicated including IGFBP4, CP, and C3 along with interferon signalling-related genes (ISG15, MX2 and OASL2). Results (Relative expression) are presented as means ± SEM from three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (ANOVA with post hoc Tukey test).

Article Snippet: Guide RNA lentiCRISPR v2 constructs targeting STAT1, STAT3, cGAS, STING and Non-targeting (NT) control were purchased from Genscript (Piscataway, NJ, USA) and generated as described previously [ ].

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Antioxidants

Article Title: Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

doi: 10.3390/antiox10020334

Figure Lengend Snippet: Model of STAT1 and STAT3 regulation of inflammation and growth arrest phenotypes in senescent preadipocytes. This schematic diagram represents how STAT1 and STAT3 function antagonistically in regulating cGAS-STING mediated induction of CXCL10 and interferon signaling related genes and p53/p21-dependent growth arrest. STAT1 cooperates with cGAS/STING in driving the induction of CXCL10, and interferon signaling related genes such as IFNα, IFNβ, ISG15, MX and OASL2 and antagonizes the function of STAT3 in inhibiting p53-dependent growth arrest and cGAS-dependent induction of interferon signaling related genes.

Article Snippet: Guide RNA lentiCRISPR v2 constructs targeting STAT1, STAT3, cGAS, STING and Non-targeting (NT) control were purchased from Genscript (Piscataway, NJ, USA) and generated as described previously [ ].

Techniques: