Fig 1. Identification of tumor suppressor genes in immortalized human bronchial epithelial BEAS-2B cells.

(A) A schematic diagram of a genetic screen for transformation suppressors of BEAS-2B cells. Retroviruses carrying the shRNA library were generated by transfection of viral packaging cells with shRNA plasmids. BEAS-2B cells were infected with FF2 shRNA or shRNA library viruses and cultured in soft agar in 10cm plates for 3 weeks, large and single colonies were isolated for cell expansion, PCR amplification and DNA sequencing of the integrated shRNAs. (B) Representative images of colonies formed on soft agar plates. Transformed colonies were observed by crystal violet staining (upper panel) or under light microscopy at 20x magnification (lower panel). (C) More and larger anchorage independent colonies were formed in library infected cells than in shFF2 infected cells, data are presented as average colony number ± standard deviation per plate based on three independent infection experiments, *p<0.05 by the Student’s t-test.