ICSBP and the type I IFNs regulate expression of the CCL6 and CCL9 chemokines. (A) Cluster analysis of the genes that changed at least 3-fold in expression among pairwise comparisons of the 4 indicated cell types: parental BaF3 cells, or BaF3 cells expressing BCR-ABL (BaF3 BA), ICSBP, or ICSBP and Bcr-Abl (ICSBP BA). Expression was normalized to that in parental BaF3 cells. Four clusters containing genes that reflect ICSBP-regulated expression are illustrated. The colors signify the magnitude and direction of the changes in gene expression. To the right of the clusters are the gene functional categories enriched in each and examples of genes present in each category. The numbers in parentheses indicate the number of times each gene appeared in a cluster. (B) RT-PCR validation of gene expression for several of the genes detected by gene expression profiling within the indicated cell types. (C) Confirmation by RT-PCR that genes regulated by ICSBP are also regulated by treatment with 10⁴ U/mL IFN alpha or beta for 6 hours in the indicated cell types. In some cases the IFNs restore expression of a gene that is otherwise down-regulated by BCR-ABL. (D) Requirement of ICSBP for IFN alpha to induce CCL6 and CCL9 expression. RT-PCR was used to detect expression of indicated genes in BaF3 parental cells expressing a control shRNA or an shRNA that decreases the expression of ICSBP. Cells were analyzed before and after treatment with IFN alpha (10⁴ U/mL for 6 hours). (E) RT-PCR showing CCL23 and CCL15 expression in the human AR230 CML cell line upon treatment with 10⁴ U/mL IFN alpha. (F) RT-PCR showing CCL23 and CCL15 expression in normal human PBMCs upon treatment with 10⁴ U/mL IFN alpha for 1 hour.