GM-CSF mediates BCR/ABL-independent IM and NI resistance. (A) Cytokine array. Different cytokines (120) were screened for differential expression in the CM of 25UR (left 2 membranes) versus 25R (right 2 membranes). Different cytokines (60) are spotted in duplicate on each membrane. Positive controls (4 dots) are shown in the top left corners. Darker spots indicate higher expression. Cytokines with increased expression in 25R-CM (bold-lined squares) compared with 25UR-CM (thin-lined squares) are indicated. (B) The viability of parental LAMA cells after 24 hours of treatment with NI in the presence of 25UR-CM and GM-CSF was assessed relative to the control treatment (no NI or GM-CSF) using the MTS colorimetric assay. Boxes display data points located in the middle 2 quartiles of all data points. Lines in boxes indicate medians; whiskers extend to the 2 extreme values of all data points. Data points of 3 independent experiments are depicted. (C) Reversal of 25R-CM–mediated NI resistance of parental LAMA cells by addition of increasing concentrations of neutralizing anti–human GM-CSF antibodies as indicated. Data points of 3 independent experiments are depicted. Bars and lines are as in panel B. (D) IM-naive parental LAMA cells were treated with NI and increasing concentrations of GM-CSF as indicated in the presence of 25R-CM or 25UR-CM. After 24 hours of treatment, cells were harvested and cell lysates were separated using PAGE and blotted with the indicated antibodies. Actin served as loading control.