Effects of the NL and RUX combination on JAK2/STAT5 signaling. CML CD34⁺ samples (n = 3) were either left untreated (UT), or treated with NL (5 µM) or RUX (200 nM) or their combination. After 24 hours of incubation in suspension cultures, p-STAT5^Tyr694 (A) and p-JAK2^Tyr1007/1008 (B) levels were measured by intracellular flow cytometry. Levels of phosphorylation of both proteins were expressed as a ratio of the mean fluorescence intensity of p-STAT5^Tyr694 (A) and p-JAK2^Tyr1007/1008 (B) antibody stained cells over the mean fluorescence intensity of cells stained with a matched isotype control. The average of UT values was normalized to 100% and changes following treatment expressed as % change from UT. (C) Candidate STAT5 target genes mRNA expression changes were measured in CML CD34⁺ samples (n = 5) following 8 hours in suspension culture with NL (5 µM) or RUX (1000 nM), or their combination. Differences in gene expression levels following treatment were calculated using the 2^−ΔΔCt method after normalization within each sample of candidate gene expression levels against the expression levels of the reference genes (GAPDH and TBP). Relative quantitation of candidate genes mRNA expression following NL, and NL and RUX treatment, was then plotted as log₂ of the 2^−ΔΔCt values (with the RUX-treated cells having a value of 0 in the graph and being the calibrator). All data from independent experiments are presented as mean ± SEM. Significance values: *P < .05; †P < .01; ‡P < .001. ns, not significant.