Fig. 3

GM-CSF/DNMT1 mediated activation of JAK2/STAT5 signaling contributed to resistance in BM microenvironment. A Cytokine array. Different cytokines (80) were screened for differential expression in monolayer culture (up membrane) versus co-culture model in K562 CD34⁺ cells (low membrane). Different cytokines are spotted in duplicate on each membrane. Darker spots indicate higher expression. Positive controls (4 dots) are shown in the top left corners. Cytokines with increased expression are indicated by bold-lined squares in co-culture system versus thin-lined squares in monolayer culture. B The survival rate of K562 CD34⁺ cells after treatment with IM for 36 h in monolayer culture and GM-CSF was evaluated comparing with the control treatment (no GM-CSF) appling MTT assay. ***p < 0.001 versus control group. Data points of triplicate experiments are depicted. C Reversal of microenvironment-mediated IM resistance of K562 CD34⁺ cells by addition of increasing concentrations of neutralizing anti–human GM-CSF antibodies as indicated. *p < 0.05, **p < 0.01 versus control group. Data points of 3 independent experiments are depicted. D, E K562 CD34⁺ cells or primary CD34⁺ CML cells were treated with IM and the raise of GM-CSF dose as indicated in BM microenvironment or in monolayer culture. After treatment for 24 h, cell lysates were acquired for western blot with the indicated antibodies. GAPDH was regarded as loading control. F, G Supplementation of anti-human GM-CSF antibodies in co-culture system, DNMT1 expression was examined in K562 CD34⁺ cells or primary CD34⁺ CML cells. H, I K562 CD34⁺ cells or primary CD34⁺ CML cells were treated with IM, then GM-CSF or α- rhGM-CSF antibodies was added into BM microenvironment or monolayer culture, respectively. The activity of DNMT1 was detected by ELISA. *p < 0.05, **p < 0.01 versus CML CD34⁺ cells treated with IM in monolayer culture. ^#p < 0.05, ^##p < 0.01 versus CML CD34⁺ cells treated with IM in co-culture system