Fig. 2

JAK2/STAT5 signaling and downstream protein were observed in CML CD34⁺ cells in BM microenvironment. BM microenvironment caused a BCR/ABL-independent activation of JAK2/STAT5 in the presence of IM in CML CD34⁺ cells. K562 CD34⁺ cells and primary CD34⁺ CML cells were cultured with or without hBMSCs for 12 h and then treated with various concentrations of IM (0, 0.125, 0.25, 0.5 μM) for 36 h, respectively. A, B Western blotting showed that activated BCR/ABL kinase was presented by staining for the expression of p-BCR/ABL and p-CrkL. STAT5 activation was shown by p-STAT5^Tyr694-specific antibodies. GAPDH was regarded as loading control. The percentages of proteins p-BCR/ABL, p-CrkL and p-STAT5^Tyr694 were observed by Western blot analysis in CML CD34⁺ cells. C, D The expressions of p-JAK2 ^Tyr1007/1008, JAK2, XIAP, Mcl-1, CyclinD2, Survivin and Bcl-2 were determined using Western blot in CML CD34⁺ cells, respectively. E, F After IM treatment, the expression of pro-apoptotic proteins caspase3, cleaved-caspase 3 and Bax were determined by Western blot in K562 CD34⁺ cells and primary CD34⁺ CML cells. *p < 0.05, **p < 0.01, ***p < 0.001 the group treated with IM in monolayer culture versus the group treated with IM in co-culture system