![Figure 1. AP-1 transcription factor activity is increased in NPM-ALK–containing cells. (A) Hierarchic clustering using gene expression data of 84 genes differentially expressed in NPM-ALK–negative (blue) and NPM-ALK–positive (red) ALCL cell lines. The dendrogram at the right demonstrates clustering of cell lines using complete linkage. (B) Hierarchic clustering using mRNA expression of 6 AP-1 target genes separating NPM-ALK–negative (blue) and NPM-ALK–positive (red) ALCL cell lines. (C) EMSA of NPM-ALK–negative (MAC-2A, FE-PD) and–positive (SR-786, Karpas 299) ALCL cell lines. Cell lysates were incubated with a [32P]-labeled AP-1 consensus DNA probe (A) or a [32P]-labeled mutated probe (M) (representative results of 3 independent experiments). (D top panel) Western blots using ALK and β-actin antibodies on lysates from vector control (VC) and NPM-ALK–expressing Ba/F3 cells, as well as TonBaF.1-NPM-ALK cells inducibly expressing NPM-ALK without (unind) and 48 hours after addition of 2 μg/mL doxycycline (+ dox). (Bottom panel) EMSA of cell lysates incubated with AP-1 consensus DNA probe (A) or mutated probe (M) (representative results of 3 independent experiments). (E top panel) Western blots of wild-type NPM-ALK and kinase-negative (KN) NPM-ALK–expressing HEK293 cells. Lysates of control (VC) and stably expressing NPM-ALK and KN transfectants were analyzed for presence of fusion protein (bottom) and for NPM-ALK phosphorylated at the pY342/pY343 position. (Bottom panel) EMSA of HEK293 KN and HEK293 NPM-ALK incubated with AP-1 consensus DNA probe (A) and mutated probe (M). (F) Supershift analysis. Cell lysates were incubated with an AP-1 consensus DNA probe and various anti–AP-1 antibodies (c-Jun, JunB, JunD, Fos, Fra-1, Fra-2, and ATF2) (representative results of 3 independent experiments). (G) JUNB mRNA and protein are increased in TonBaF.1-NPM-ALK cells induced for 48 hours with doxycycline. (Left) mRNA expression levels relative to a calibrator (c-JUN mRNA level of Mac2A cells compared with β-actin) are given to facilitate comparison between AP-1 factors (bars indicate standard deviation [SD] of triplicate experiments). (Right) Immunoblotting using antibodies indicated on lysates of uninduced and induced TonBaF.1-NPM-ALK cells.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/9/10.1182_blood-2007-02-071258/7/zh80230709630001.jpeg?Expires=1736053688&Signature=cGwVmVr9u4UofEXnOZDOJtFHYKfpxA4u2SInpiH6AnPsjgQCyL10G5xf8oZQ463PrquOwK7R~D-dI7eu56iOUcgJu8xVwnQ2U9o6n6XKfz2Hk4hZ9ITrgbi~7CR0Zd2zPpcHvTRm0we7OV803PKP0UxBx235mlGZhzYkKWOKlOhapsSOMDivf4GcHJAmET-OLhSl27dBNTwg0SsfCA68CNn2hWB-POlxdcMJQb7yNxeZRTszRvZo6ycGXl1k9HX9FiQAALTWryIdTp2Xv8xyipk-73TyAERkEpciGq6H77jEs2Nh-wCyYQM~TxYYtXZv9RgshtLSeI~EO7ipsoSQZw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
AP-1 transcription factor activity is increased in NPM-ALK–containing cells. (A) Hierarchic clustering using gene expression data of 84 genes differentially expressed in NPM-ALK–negative (blue) and NPM-ALK–positive (red) ALCL cell lines. The dendrogram at the right demonstrates clustering of cell lines using complete linkage. (B) Hierarchic clustering using mRNA expression of 6 AP-1 target genes separating NPM-ALK–negative (blue) and NPM-ALK–positive (red) ALCL cell lines. (C) EMSA of NPM-ALK–negative (MAC-2A, FE-PD) and–positive (SR-786, Karpas 299) ALCL cell lines. Cell lysates were incubated with a [32P]-labeled AP-1 consensus DNA probe (A) or a [32P]-labeled mutated probe (M) (representative results of 3 independent experiments). (D top panel) Western blots using ALK and β-actin antibodies on lysates from vector control (VC) and NPM-ALK–expressing Ba/F3 cells, as well as TonBaF.1-NPM-ALK cells inducibly expressing NPM-ALK without (unind) and 48 hours after addition of 2 μg/mL doxycycline (+ dox). (Bottom panel) EMSA of cell lysates incubated with AP-1 consensus DNA probe (A) or mutated probe (M) (representative results of 3 independent experiments). (E top panel) Western blots of wild-type NPM-ALK and kinase-negative (KN) NPM-ALK–expressing HEK293 cells. Lysates of control (VC) and stably expressing NPM-ALK and KN transfectants were analyzed for presence of fusion protein (bottom) and for NPM-ALK phosphorylated at the pY342/pY343 position. (Bottom panel) EMSA of HEK293 KN and HEK293 NPM-ALK incubated with AP-1 consensus DNA probe (A) and mutated probe (M). (F) Supershift analysis. Cell lysates were incubated with an AP-1 consensus DNA probe and various anti–AP-1 antibodies (c-Jun, JunB, JunD, Fos, Fra-1, Fra-2, and ATF2) (representative results of 3 independent experiments). (G) JUNB mRNA and protein are increased in TonBaF.1-NPM-ALK cells induced for 48 hours with doxycycline. (Left) mRNA expression levels relative to a calibrator (c-JUN mRNA level of Mac2A cells compared with β-actin) are given to facilitate comparison between AP-1 factors (bars indicate standard deviation [SD] of triplicate experiments). (Right) Immunoblotting using antibodies indicated on lysates of uninduced and induced TonBaF.1-NPM-ALK cells.
