Identification of suppressors of ATRA-mediated cell growth arrest using an shRNA screening approach. (A) Soft-agar colony-formation assay. NB4 cells grew into colonies in RPMI/0.2% in the absence of ATRA. In the presence of 0.5 μM ATRA, NB4 cells stopped proliferation in soft agar and differentiated into mature neutrophils in 7 days. Cells were plated at a density of 5 × 105 cells/100-mm plate. Images were captured using a 20× dry objective (NIA 0.5) on an Olympus IX-71 microscope (Melville, NY). Images were acquired using a CCD camera (SensiCam, Cooke, Romulus, MI) controlled by IPLab imaging software (IPLab, Rockville, MD). The figure shows the results of a representative experiment. (B) The experimental flow chart of the functional shRNA screening. Details are provided in “Retroviral infection for high-throughput shRNA screening.” (C) An ATRA-resistant positive colony identified from the initial screening. This colony is GFP positive and thus contains the inserted viral sequence. The images were acquired as described in panel A.
