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1) Product Images from "SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia"
Article Title: SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia
Journal: Science Advances
Figure Legend Snippet: SF3B1 is posttranslationally regulated in T cell leukemia. ( A ) Relative essentiality of the U2 splicing complex components across different types of cancers. Essentiality data were obtained from the Project Achilles CRISPR-Cas9 screening dataset for 563 cancer cell lines. ( B ) Immunoblot showing SF3B1 protein level in patients versus T cells [see fig. S2D for the quantification of protein levels, (N1-IC, NOTCH1 intracellular domain)]. ( C ) Reverse transcription reaction coupled to reverse transcription polymerase chain reaction (RT-PCR) for the SF3B1 mRNA expression in T-ALL versus T cells in human sample ( n = 3). * P ≤ 0.05. ( D ) RPPA analysis for SF3B1 protein levels in HR ( n = 17) versus non-HR ( n = 25) patient cases. ( E ) Immunoblot indicating SF3B1 protein expression level in CUTLL1 and JURKAT cells treated with 10 μM PR619, 2 μM b-AP15, or 200 nM ML323 (24 hours). GAPDH is used as the loading control. ( F ) SF3B1 protein level upon treatment of CUTLL1 and JURKAT with 10 μM P5091 for 24 hours. ( G ) Representative coimmunoprecipitation analysis of USP7 (left) and SF3B1 (right) to evaluate the interaction between USP7 and SF3B1 in CUTLL1 cells. IP, immunoprecipitation; IgG, immunoglobulin G. ( H ) Representative immunoblot of SF3B1 protein expression upon treatment with cycloheximide (CHX) (10 μg/ml) in control - and shUSP7 -expressing RPMI-8402 cells for 12 hours. Quantification of protein levels is shown (right). ( I ) Representative immunoblot of SF3B1 protein expression upon treatment of cells with 10 μM P5091 (24 hours). ( J ) Immunoprecipitation of Flag-tagged SF3B1 coupled to immunoblot analysis for detection of SF3B1 ubiquitination in 293T cells cotransfected with hemagglutinin (HA)–tagged or HA-K63O (only K63 can be ubiquitinated) ubiquitin (Ub) construct and Flag-SF3B1.
Techniques Used: CRISPR, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Construct
Figure Legend Snippet: SF3B1 silencing or inhibition blocks growth of T cell leukemia. ( A ) Immunoblot indicating SF3B1 depletion in CUTLL1 and JURKAT cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( B ) CUTLL1 and JURKAT cells were counted at the indicated time points, and cell growth was plotted as shown for the shSF3B1#1 - and shSF3B1#2 -expressing cell populations ( n = 3). ( C ) Luciferase-expressing CUTLL1 cells were transduced with control or shSF3B1#2 and injected intravenously (retro-orbitally) into immunocompromised mice. Luminescence analysis for representative mice on days 8 and 24 (left) and luminescence intensity fold change between days 8 and 24 are shown (right: control , n = 5; shSF3B1#2 , n = 6). ( D ) Survival curve analysis of mice from (C). * P ≤ 0.05 and ** P ≤ 0.01. ( E ) E7107 treatment schema in the xenograft model. ( F ) Luciferase-expressing CUTLL1 cells were intravenously (tail vein) injected into immunocompromised mice followed by 8 days of E7107 administration (at 5 mg/kg per day) starting on the 10th day after transplantation. Luminescence images for representative mice on days 10 and 20 (left) and luminescence intensity fold change between days 10 and 20 are shown (right: vehicle, n = 9; E7107, n = 9). ( G ) Survival analysis of mice from (F). ( H ) Mouse spleen size (left) and weight (right) upon E7107 treatment of the NOTCH1-δE-Cherry + retroviral T-ALL model [vehicle ( n = 3), E7107 ( n = 5)]. ( I ) Representative mice body weight, liver weight, and spleen weight analysis upon treatment with E7107. ( J ) Representative hematoxylin and eosin (H E) staining of esophagus, stomach, jejunum, and ileum (×200 magnification).
Techniques Used: Inhibition, Expressing, Luciferase, Transduction, Injection, Mouse Assay, Transplantation Assay, Staining
Figure Legend Snippet: SF3B1 activity is critical for CHEK2 levels and DNA damage response. ( A ) γH2AX level in CUTLL1 and JURKAT cells treated with 3 nM E7107 for 24 hours. ( B and C ) Representative comet assay photos from CUTLL1 cells treated with 3 nM E7107 for 24 or 48 hours [quantification of (C) in the right panel, n = 45]. ** P ≤ 0.01. ( D ) Metagene analysis of MapR signal around the transcriptional start site in 3 nM E7107 treatment for 24 hours in CUTLL1. DMSO, dimethyl sulfoxide. ( E ) Metagene analysis of normalized transient transcriptome sequencing (TT-seq) reads for protein-coding transcripts (±3-kb area, 3 nM E7107, 15 min). ( F ) Scatterplot of splicing changes in E7107 versus vehicle (FDR
Techniques Used: Activity Assay, Single Cell Gel Electrophoresis, Sequencing