Figure 2. DDX5 is required for AML progression in vivo.

(A) Mouse LG1 AML cells were infected with virus encoding GFP and either of two different shRNAs targeting murine DDX5 (shDDX5.1322 or shDDX5.2086) or shRen.713. ShRNA expression in LG1 cells is constitutive following infection. Similar to Figure 1 WCE prepared from LG1 cells transduced with shRen.713 is loaded onto the immuno-blot either undiluted (lane 3), diluted 1-to-4 (lane 4) or 1-to-10 (lane 5) and WCE from LG1 cells transduced with either shDDX5.1322 or shDDX5.2086 are loaded in lanes 1 and 2 respectively. The immunoblot in the upper panel is detecting DDX5 protein expression and the lower panel is the Ponceau S stained membrane to show protein loading in the wells. Cell proliferation was analyzed using the competition proliferation assay as described in Figure 1. Data is presented as the mean from triplicate samples with standard deviations shown. (B) Clonal RN2 AML cells selected for either doxycycline-induced shDDX5.2086 or shRen.713 shRNA expression were transplanted by tail vein injection from leukemic spleens into secondary recipient mice as described in Experimental Procedures. The AML cells constitutively express Firefly luciferase enabling bioluminescence imaging of the transplanted leukemia cells in vivo. Day 0 indicated to the left of the bioluminescence imaging results indicates the time-point 5 days after bone marrow transplantation with AML cells when leukemia was established in the mice and they were separated into either doxycycline treated or untreated groups. (C) Kaplan-Meier curves showing survival of mice transplanted with AML cells either induced or not induced to express the indicated shRNAs. Time zero on these plots indicates the time-point when the AML cells were transplanted into the mice. Doxycycline was begun on day 5 post-transplantation as indicated by the green arrow.