Figure 6.

A. Viability of NSCLC cell line A549 is inhibited by RB-011 and RB-012 as determined by MTS assay after 48 h treatment. Error bars represent standard error of triplicate measurements. B. RB-011 and RB-012 induce caspase-3 activation at 48 h in A549 cells. The error bars show the range of duplicate determinations: and the results are representative of several experiments. C. Effect of RB-011 and -012 on A549 colony growth in soft agar. Results are expressed relative to colony numbers in untreated controls. D. Immunoblotting analysis of phospho-ERK over time induced by 25 μM RB-012 treatment of A549 cells. E. Growth of A549 xenograft in BALB/c nude mice is retarded by administration of RB-012. RB-012, saline or FTY720 was administered daily to mice bearing A549 tumors by intraperitoneal injection using the dosing regime shown. All experimental data are shown as the mean ± SEM. ** indicates P < 0.05. F. RB-012 induces down-regulation of MAPK signaling in A549 xenografts. Tumors were excised at the end of the study and analyzed by immunofluorescence for phospho-ERK. Area coverage analysis is represented by a box and whisker plot. Statistical significance was assessed using the Mann-Whitney test and Dunnet's post hoc test, ** indicates P < 0.05 for N = 7 samples with multiple fields analyzed. Representative images of phospho-ERK immunofluorescence are shown below, scale bar – 100 μm.