Elevated STAT5 levels are found in advanced CML phases, and STAT5 down-regulation increases imatinib response of human CML cell lines. (A) RT-PCR of human STAT5A mRNA transcripts of BM derived from healthy control patients (n = 3), PB derived from untreated CML patients in CP (n = 13), AP (n = 7), and relapsed imatinib-resistant patients (n = 6 for CP, one in AP, and one in BC). CML phase, kind of relapse (C indicates cytogenetic; H, hematologic; and M, molecular), BCR-ABL1 mutations, IS value for BCR-ABL1 mRNA level, and fold change STAT5A mRNA levels compared with mean STAT5A mRNA level of imatinib-sensitive CP samples are indicated. nd indicates not determined. Each bar represents data derived from an individual patient. Results were normalized by comparison with their GAPDH mRNA expression. P values for patients in imatinib-sensitive CP compared with imatinib-sensitive AP and imatinib-resistant CP are indicated. (B) STAT5A/B immunoblot of BM derived from a healthy patient (control BM) and PB derived from CML diseased patients in CP (n = 6), AP (n = 3), and imatinib-resistant CP (n = 3). Each lane represents an individual patient. (C) Expression of pSTAT5 in primary CML cells. Mononuclear BM cells from 3 CML patients, one in CP (top image), one in AP (middle image), and one in BC (C, bottom image), were spun on cytospin slides and stained with anti-pSTAT5 antibody AX1. Subsets of CML cells in AP and BC were found to stain positive for pSTAT5, whereas fewer cells expressed pSTAT5 in CP. Representative examples are shown. (D) RT-PCR for STAT5A mRNA from total MNCs and CD34⁺/lin⁻ sorted MNCs derived from 3 individual CML patient samples. (E) KU812 and (F) K562 cells expressing siSTAT5 or siRDM were treated 48 hours with imatinib. Shown is the relative decrease of siSTAT5⁺ compared with siRDM⁺ cells. Percentages of siRDM expressing cells served as baseline and were set to 100%.