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Results: 6

1.
Figure 1

Figure 1. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Expression of TAK1 in lymphoma cell lines. (A) Lymphoma cell lines have greater expression of TAK1 and pTAK1 compared with PBMCs from healthy donors. TRAF6, which is frequently required for TAK1 activation, and β-actin were used as controls. (B) Immunohistochemical analysis of TAK1 and pTAK1 expression in the cytospins of MCL lines demonstrating a cytoplasmic distribution pattern for pTAK1.

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.
2.
Figure 3

Figure 3. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Identification of the small molecule inhibitor AZ-TAK1. (A) Docked pose of AZ-TAK1 in TAK1 crystal structure. Binding mode of AZ-TAK1 (1-(2-(3-ethyl-5-(5-fluoro-4-(imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-ylamino)phenoxy)ethyl)piperidin-4-ol) is consistent with in house cocrystal structures of close analogs of AZ-TAK1 (Proteros Biostructures GmbH, unpublished work, August 10, 2008). AZ-TAK1 adopts a U-shaped conformation in TAK1, with the imidazopyridazine heterocycle packing atop C148 and aminopyrimidine making 2 hydrogen bond interactions with the hinge (A81). The 3-ethyl moiety points toward the sugar pocket and the 4-hydroxypiperidine is out in the solvent channel. (B) The ability of AZ-TAK1 to inhibit TAK1 kinase activity was measured in vitro with the use of a complex of recombinant TAK1 and TAB1 and the substrate MKK6kd. Data shown are representative of 3 independent assays. (C) Selectivity of AZ-TAK1 against a panel of 30 kinases.

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.
3.
Figure 6

Figure 6. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Effect of AZ-TAK1 on primary lymphoma cells. (A) TAK1 and pTAK1 are abundantly expressed in primary MCL cells. Incubating the cells with 300nM of AZ-TAK1 for 24 hours inhibited TAK1 phosphorylation, down-regulated XIAP, and cleaved caspase 3. (B) TAK1 expression in lymph node sections of primary MCL, classic HL, DLBCL, and ALK (+) ALCL cases. Sections of an infiltrating ductal breast carcinoma and a reactive lymph node (LN) were used as positive and negative controls, respectively. Representative staining of negative case (blastoid type), moderately positive (+), and strongly positive (++) MCL cases are shown. (C) Summary of primary cases expressing pTAK1 by IHC (D) Primary lymphoma cells were incubated with 300nM of AZ-TAK1 for 24 hours before cell viability was determined by the use of the MTS assay. Results are the mean of 3 experiments (± SEM).

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.
4.
Figure 2

Figure 2. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Biologic and molecular consequences of silencing TAK1 expression in lymphoma. (A) A representative experiment of TAK1 gene silencing with the use of 3 different siRNAs in lymphoma cell lines. TAK1 protein levels were determined by Western blot after 48 hours of transfection. Similar results were obtained in 2 additional independent experiments. (B) TAK-1 gene silencing decreased the phosphorylation of p38 and IκBα without affecting ERK phosphorylation. Results are shown after 48 hours of transfection. (C) TAK1 gene silencing resulted in induction of apoptosis in lymphoma cell lines, as determined by the annexin V/propidium iodide staining method and FACS analysis. This is a representative experiment performed after 48 hours of transfection with TAK1 siRNA or control siRNA. (D) Summary results of TAK1 siRNA–induced cell death. Each value is the mean of 3 independent experiments performed in triplicate (± SEM). **P < .005. Open bars indicate RPMI; gray bars, control siRNA; and black bars, TAK1 siRNA3.

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.
5.
Figure 4

Figure 4. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Molecular and biologic consequences of pharmacologic inhibition of TAK1 in lymphoma cell lines using AZ-TAK1. (A) Cell lines of Hodgkin lymphoma origin (HD), MCL, or anaplastic large cell lymphoma (ALCL) were incubated with increasing concentrations (0.1, 0.5, 1, and 2μM) of the AZ-TAK1 for 72 hours before cell viability was determined with the MTS assay. PBMCs from 3 healthy donors were included for comparison. Each value is the mean of 3 independent experiments performed in triplicate (± SEM). (B) IC50 values for AZ-TAK1 in lymphoma cell lines and PBMCs at 72 hours (C) Effect of AZ-TAK1 on induction of apoptosis. The MCL cell lines Jeko-1, Mino, and SP53 were incubated with dimethyl sulfoxide (DMSO; 0.1%) or AZ-TAK1 (0.1 or 0.5μM) for 48 hours before apoptosis was measured using the annexin V/PI dual staining method and FACS analysis. (D) Summary results of AZ-TAK1–induced cell death (PI and annexin V–positive cells) from 3 independent experiments. Each value is the mean of 3 independent experiments performed in triplicate (± SEM). **P < .005. (E) Effect of AZ-TAK1 on cell-cycle analysis. The MCL cell lines Jeko-1, Mino and SP53 were incubated with DMSO (0.1%) or AZ-TAK1 (0.1 or 0.5μM) for 24 hours before cell-cycle analysis was performed using the PI staining method by FACS analysis.

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.
6.
Figure 5

Figure 5. From: Essential role of TAK1 in regulating mantle cell lymphoma survival.

Molecular mechanisms of AZ-TAK1 antiproliferative activity lymphoma. (A) MCL cell lines were incubated with medium or 0.5μM of AZ-TAK1 for 24-48 hours. Whole-cell lysates were examined by Western blot for changes in intracellular proteins. AZ-TAK1 decreased TAK1 and p38 phosphorylation, indicative of their inactivation. AZ-TAK1 inactivated NF-κB, as indicated by the decrease in nuclear p65 and inhibition of IκB phosphorylation. (B) AZ-TAK1 down-regulated XIAP and activated caspase 9 and caspase 3. (C) AZ-TAK1–induced cell death was either partially or completely blocked by the caspase 9 inhibitor Z-LEHD-FMK. Each value represents a mean of 3 independent experiments done in triplicate (± SEM). (D) AZ-TAK1 released cytochrome c and SMAC/Diablo from the mitochondria in MCL cell lines (top). Western blot analysis was performed on subcellular mitochondrial fractions after incubation with AZ-TAK1 (0.5μM) for 48 hours. Cytofluorimetric analysis of MCL cell lines mitochondrial potential, as assessed with the mitochondrial specific cationic dye (middle), as described in “Evaluation of mitochondrial membrane potential.” In this representative experiment, treatment with AZ-TAK resulted in mitochondria depolarization in 75% of the cells, compared with control 8% in DMSO-treated cells. Summary of 3 independent experiments demonstrating the effect of AZ-TAK1 on mitochondria membrane polarization (bottom). Each value represents the percentage of cells with depolarized mitochondria. Cells were incubated for 12 hours in the absence (DMSO) or presence of AZ-TAK1 0.5μM.

Daniela Buglio, et al. Blood. 2012 July 12;120(2):347-355.

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