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. Author manuscript; available in PMC: 2016 May 12.
Published in final edited form as: Leukemia. 2014 Dec 23;29(6):1390–1401. doi: 10.1038/leu.2014.347

Figure 3. TRAF1-ALK leads to the constitutive activation of NFkB transcription members.

Figure 3

Figure 3

(A–B) Lysates from a representative NPM-ALK cell line (SUP-M2 and Karpas 299) and leukemic ALCL-11 cells were immune-precipitated with an anti-ALK (Panel A) and anti-TRAF1 (B) antibodies. Immuno-complexes were resolved and then blotted with the indicated antibodies. Immuno-precipitates in presence of mouse serum are depicted as well (Beads). TL: Total Lysates, SN: Supernatant and IP Immune Precipitation. Protein molecular weights are shown. (C) Lysates from HEK293T cells, transfected with the full length TRAF1-ALK and ΔTRAF1-ALK, were immuno-precipitated with an anti-TRAF2 antibodies. Immuno-complexes were resolved and blotted with anti-TRAF1 antibodies. The expression of the corresponding proteins prior the immune-precipitation is shown (Total lysate) Predicted protein molecular weights are indicated.

(D) ALCL-11 cells show constitutive activation of the NFkB pathway. Primary ALCL-11 cells were immune-blotted with antibodies recognizing p105/p50 and p100/p52 NFkB. NPM-ALK+ ALCL cell lines were used as control.

(E) Luciferase Expression of transfected HEK-293T cells. Cells were transfected with the indicated cassettes expressing CD30, NFkB-ROS, CD30+NPM-ALK (active form), CD30+K210R NPM_ALK (inactive form) and CD30+ EML4-ALK.

(F) Knock-down of p50 or p52 impairs the NFkB mediated luciferase expression via CD30 signaling. HEK-293T cells were transfected with the indicated cassettes in presence of a CD30 expression vector.

(G) Protein expression of untreated and treated (Bortezomib 5nM/10nM) ALCL-11-PDTs was determined at 36 hr by Western Blotting with specific antibodies.

(H) Panel shows the normalized levels of mRNA expression of NFkB-regulated genes in untreated (control) or Bortezomib treated ALCL-11 leukemic cells (10nM, at 6 and 12 hr of culture). Expression levels were determined using a qRT-PCR approach as previously described {Piva, 2010 #8623;Agnelli, 2012 #599}. Data are depicted as 2^ddCt. Expression levels GAPDH of 6 hr treated (reference value) and 12 hr treated cells are reported.

(J) ALCL-11 cells are sensitive to Bortezomib. Primary cells (1×105/ml) were treated with increasing dose of the drug, overtime. Data have been normalized to control DMSO treated cells. DMSO viability decreased over time with a 30–40% spontaneous cell death at 36hrs.