Fig. 5.

Knockdown of kinases results in growth in soft agar. (A) Knockdown of IRAK4 and NME1 promotes growth in soft agar. HBE135 cells were transduced with scrambled shRNAs or two shRNAs targeting IRAK4 or NME1. sh, short hairpin. Cells were selected for 21 d, seeded in soft agar, and incubated for an additional 21 d. Colonies were stained with crystal violet. (B) Soft agar growth requires sustained knockdown of NME1. HBE135 cells were transduced with NME1 shRNA and plated in a soft agar matrix at 4 or 17 d postinfection. The colony number is the average of three (4 d) or two (17 d) wells ± SEM. (C) Soft agar colonies are aneuploid. A single colony was picked from soft agar in cells containing the NME1 shRNA. The colony was expanded for several passages, and the cells were fixed for FISH analysis as described in Fig. 4B.