Fig. 2. Efficacy of CREB Inhibition Depends on CREB Expression.

A) IC₅₀ values for 4 AML cells lines identically treated with XX-650-23 are shown. Cells were cultured with various doses of XX-650-23 for 48 hours. Viable cells were enumerated by Trypan blue exclusion method. B) Western blot of CREB expression levels in 4 AML cell lines, run on non-contiguous lanes. C) Six AML patient and three normal bone marrow samples were treated with 2 μM XX-650-23 for 48 hours, and the percent of viable cells lost or gained compared to DMSO-treated cells is shown. Data are graphed as mean ± SEM (n = 4). D) Western blot of CREB expression in ten AML patient and four normal marrow samples. Relative expression levels of CREB protein are shown. E) Methylcellulose colony assays of normal bone marrow progenitor cells treated with up to 10 μM XX-650-23. Cells were cultured with XX-650-23 for 2 weeks in methylcellulose media and colonies were scored based on morphology. Data are graphed as mean ± SEM (n = 3). F) Western blot of CREB expression levels in KG-1 cells engineered to overexpress CREB (CREB OE) or GFP control (GFP), or in which CREB expression was reduced by shRNA (CREB KD). Representative blots of at least three independent experiments are shown. G) XX-650-23 dose-response data for the three KG-1 cell lines depicted in (F) following 48 hours of treatment.