Fig. 3.

Both AZD1208 treatment and PIM2 knockdown act additively to block Raji cells proliferation. a-d Cells from two different single cell clones expressing shPIM2 were incubated with or without 5 mM IPTG for two days before drug treatment and then maintained throughout with 5 μM AZD1208 or DMSO used as vehicle control. In absence of IPTG, shPIM2 was not expressed. Media was changed and fresh drug/IPTG added every 2 days. a Western blots showing knockdown of PIM2 at day 6, after treatment with or without 5 mM IPTG and DMSO (d) or AZD1208 (a). b Viable cell counts using trypan blue. The means and SD of 2 independent experiments conducted in triplicate wells of six-well plates are shown. c Analysis of clone 1 cell viability at day 6, using Annexin V and propidium iodide (PI) staining as measured by flow cytometry and expressed in % viable and apoptotic cells. No change in the percentage of apoptotic cells is observed with clone 2. d Cell cycle analysis: cellular DNA was stained with PI and the percentage of cells in G0/G1, S and G2/M phases measured by flow cytometry. For each clone, the experiment has been repeated twice. Paired student’s t test comparing no IPTG versus IPTG is indicated at the bottom of the figure. This t test is not significant when comparing DMSO and AZD1208 treatments