Role of IFNAR1 down-regulation in CML cell viability and clonogenicity. (A) KT1 cells were transfected with control shRNA or shPKD2 and cotransfected with plasmid encoding hygromycin resistance. The cells were kept under hygromycin selection for 48 hours, and viability in response to IFNα was assessed by trypan blue staining and depicted as relative values. (B) KT1 cells were transfected with empty vector, plasmid expressing wild-type IFNAR1, or IFNAR1^S535A. The cells were cotransfected with plasmid encoding hygromycin resistance. The hygromycin-resistant cells were selected for 48 hours and analyzed for their viability after IFNα treatment as described in panel A. (C) Colony formation assays were carried out with CML samples from peripheral blood of patients in the chronic phase (773, 894, and 954) and blast crisis (623 and 967) who were positive for Bcr-abl expression. Cells were preincubated with either vehicle, IM (0.25μM), or IFNα (1000 IU/mL) for 6 hours followed by incubation with IFNα (1000 IU/mL) for 24 hours. Cells were then washed and plated in drug-free methylcellulose media, and the colonies were scored 2 weeks later and depicted as percentage relative to untreated controls. (D) Colony formation assays were carried out with CML samples from peripheral blood of patients in the chronic phase (62, 383, 586, 845, 852, 1000, 1067, 1276, 1953, and 2299) and blast crisis (834 and 1420) who were positive for Bcr-abl expression. Cells were preincubated with either vehicle, IM (0.15μM), or IFNα (150 IU/mL) for 6 hours followed by incubation in the methylcellulose media containing or not IFNα (150 IU/mL) for 3 weeks before the colonies were scored and depicted as percentage relative to untreated controls. Asterisks denote statistically significant (compared with other treatment groups) differences, *P < .05 and **P < .001.