Journal: Cancer Cell International

Article Title: ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma

doi: 10.1186/s12935-019-0754-9

Figure Lengend Snippet: Phosphorylated ITK expression in AITL patients and malignant T cell lymphoma cell lines. a Immunohistochemical staining analysis of the expression of phosphorylated ZAP70, ITK and PLCγ1 in paraffin-embedded tumor tissues from AITL patients and representative pictures. Each sample was graded independently by two pathologists. b , c Kaplan–Meier curve of PFS and OS according to the expression of phosphorylated ITK. d , e ITK and phosphorylated ITK expression level in malignant T cell lymphoma cell lines were analyzed using real-time PCR and Western blot. Jurkat, Hut-78 and H9 cells expressed total ITK and phosphorylated ITK as a band at 72 kDa. GAPDH is shown as a loading control (36 kDa). Data are expressed as Mean ± SD and representative of three independent experiments

Article Snippet: Antibodies against phospho-ZAP70 (Tyr 493, 1:100, LifeSpan BioSciences, Seattle, WA, USA), phospho-ITK (Tyr512, 1:50, LifeSpan BioSciences, Seattle, WA, USA), and phospho-PLCγ1 (Tyr783, 1:500, Cell Signaling Technology, Danvers, CA, USA) were used for immunohistochemical analysis.

Techniques: Expressing, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Cell International

Article Title: ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma

doi: 10.1186/s12935-019-0754-9

Figure Lengend Snippet: Characteristics of variants identified by whole exome sequencing of TCR-related genes in 12 AITL samples

Article Snippet: Antibodies against phospho-ZAP70 (Tyr 493, 1:100, LifeSpan BioSciences, Seattle, WA, USA), phospho-ITK (Tyr512, 1:50, LifeSpan BioSciences, Seattle, WA, USA), and phospho-PLCγ1 (Tyr783, 1:500, Cell Signaling Technology, Danvers, CA, USA) were used for immunohistochemical analysis.

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: Cancer Cell International

Article Title: ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma

doi: 10.1186/s12935-019-0754-9

Figure Lengend Snippet: ITK inhibition reduced proliferation and inhibited TCR signaling in malignant T cells. a Jurkat, Hut-78, and H9 cells were transfected with vectors carrying ITK shRNA (shITK-34465, shITK-34466 and shITK-34467) or a nonspecific shRNA (shControl). Viable cells were analyzed using the Cell Titer-Glo Luminescent Cell Viability Assay. b Jurkat, Hut-78, and H9 cells transfected with shITK or shControl were subjected to Western blotting analysis of p-PLCγ1, p-Akt, Akt, p-Erk, and Erk. GAPDH is shown as a loading control. c Karpas-299 cells were transfected with carrying ITK shRNA (shITK-34465, shITK-34466 and shITK-34467) or a nonspecific shRNA (shControl). Viable cells were calculated using the Cell Titer-Glo Luminescent Cell Viability Assay. d Jurkat, Hut-78, H9, Karpas-299 and T cells from healthy donors were treated with the indicated concentrations of the ITK inhibitor BMS-509744. Viable cells (% control) were calculated. e Jurkat, Hut-78, and H9 cells were pretreated with BMS-509744 (3 μM, 5 μM, or 8 μM) for the indicated times (60 min, or 90 min), and the whole-cell extracts were subjected to Western blot. f Jurkat, Hut-78, and H9 cells were pretreated with BMS-509744 (3 μM, 5 μM, or 8 μM) for 24 h, and the whole-cell extracts were subjected to Western blot. Data are expressed as Mean ± SD and representative of three independent experiments. Data are expressed as Mean ± SD and representative of three independent experiments

Article Snippet: Antibodies against phospho-ZAP70 (Tyr 493, 1:100, LifeSpan BioSciences, Seattle, WA, USA), phospho-ITK (Tyr512, 1:50, LifeSpan BioSciences, Seattle, WA, USA), and phospho-PLCγ1 (Tyr783, 1:500, Cell Signaling Technology, Danvers, CA, USA) were used for immunohistochemical analysis.

Techniques: Inhibition, Transfection, shRNA, Cell Viability Assay, Western Blot

Journal: Cancer Cell International

Article Title: ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma

doi: 10.1186/s12935-019-0754-9

Figure Lengend Snippet: ITK inhibition induced apoptosis and G2/M phase arrest in malignant T cell lymphoma cells. a , b Jurkat, Hut-78 and H9 cells (2 × 10 5 ) were treated with BMS-509744 (3 μM, 5 μM, or 8 μM) for 24 and 48 h, and apoptotic cells were quantified using flow cytometry. c Hut-78, H9 and Jurkat cells (2 × 10 5 ) were treated with BMS-509744 (3 μM, 5 μM, or 8 μM) for 48 h. Whole-cell extracts were subjected to Western blot using antibodies directed against caspase-3, PARP, Mcl-1, Bcl-xl, Bcl-2, and Bak. GAPDH is shown as a loading control. d , f Jurkat, Hut-78 and H9 cells (2 × 10 5 ) were treated with different concentrations of BMS-509744 (3 μM, 5 μM, or 8 μM) for 24 h, and the cell cycle profiles of the populations were measured using flow cytometry. e Western blot was used to quantify the expression of cell cycle-related proteins (p-cdc2, cdc2 and cyclin B1) after treatment with different concentrations of BMS-509744 (3 μM, 5 μM, or 8 μM) for 48 h. GAPDH is shown as a loading control. Data are expressed as Mean ± SD and representative of three independent experiments. Statistical analysis was performed using Student’s t test. * P < 0.05, ** P < 0.001 compared with control group

Article Snippet: Antibodies against phospho-ZAP70 (Tyr 493, 1:100, LifeSpan BioSciences, Seattle, WA, USA), phospho-ITK (Tyr512, 1:50, LifeSpan BioSciences, Seattle, WA, USA), and phospho-PLCγ1 (Tyr783, 1:500, Cell Signaling Technology, Danvers, CA, USA) were used for immunohistochemical analysis.

Techniques: Inhibition, Flow Cytometry, Western Blot, Expressing