Figure 2.

NPM1-ALK Activates STAT3 Pathway and Increases Cell Growth in Low-Serum Culture

(A) In human cells, NPM1 and ALK genes are located on chromosomes 5 and 2, respectively. To induce t(2; 5) translocation, CRISPR/Cas9 system is used to induce specific NPM1 (using sgRNA^NPM1) and ALK (using sgRNA^ALK) DSBs. The derivative chromosome (Der5) will lead to NPM1-ALK fusion gene expression. Der5 and Der2 junctions were detected in the four clones Lenti-CL1 (obtained using a lentivirus vector), CLA, CLB, and CLC isolated from sgRNA^NPM1- and sgRNA^ALK-treated cells.

(B) FISH for NPM1 and ALK detection in Lenti-CL0 (control clone) and Lenti-CL1 metaphases. ALK locus is detected with a break-apart probe (green + purple). Blue signal corresponds to the 5′ end NPM1 locus: Der5 and Der2 formation leads to split purple and green signals. Colocalization of purple and blue signals reveals Der5 formation.

(C) Western blot detection of NPM1-ALK and STAT3 activation in the translocated clones. SUDHL1 is an ALK-positive ALCL cell line. Lenti-CL0 and Lenti-CL1 were also treated with different amounts of crizotinib.

(D) Upper panel: growth curve of wild-type RPE-1 cells and Lenti-CL1 in 10% FBS. Lower panel: competitive proliferation assay between wild-type RPE-1 cells and Lenti-CL1 in the presence of 10% or 2% FBS. Lenti-CL1 stably expressed GFP (mean of three experiments ± SD).

(E) FISH analysis of RPE-1 clones growing in 2% FBS: CLA, CLB, and CLC. A break-apart probe (purple and green) was used for ALK and a BAC probe (blue) was used to probe NPM1. Der5 and Der2 formation leads to split purple and green signals. Colocalization of purple and blue signals reveals Der5 formation.

See also Figure S2.