Figure 5.
SET2-S cell evaluation in vitro and in vivo. Error bars: 5th to 95th percentile. BLI imaging and growth curves use exponential scale. (A) Western blot evaluating the protein expression of p27Kip1 measured using a FLAG specific antibody. (B) Dose-dependent growth inhibition of SET2-S cells in vitro with increasing doxycycline concentrations. (C) Competition assay demonstrating decrease in population of SET2-S cells when compared to SET2 cells. SET2 and SET2-S cells were mixed 50:50 at t = 0 h and 2 µg/mL doxycycline was added to the media. Expression of tdTomato, marking SET-S cells, was measured over time. (D) BLI at the end of 3 weeks of therapy in NSG mouse model of SET2-S leukemia (day 28 status-post leukemic injection). Controls had injections of SET2-S cells and no therapy. ESKM treated mice received the antibody only on day 6 onwards. The other two groups got doxycycline alone or doxycycline with ESKM. (E) A plot on log scale of the mice treated in panel D over one month. SET2-S in vivo growth, by BLI, evaluating ESKM ADCC with and without p27Kip1 overexpression, demonstrating clear superiority of ADCC treatment on slower growing SET2-S cells compared to wild type controls. (F) Flow cytometry of SET2-S cells with and without doxycycline exposure, evaluating ESKM binding. (G) ADCC of SET2-S with and without upregulation of p27Kip1, activated by doxycycline exposure. The overexpression of p27Kip1 appears to stabilize SET2-S, decreasing the chromium release of both control and ESKM treated cells.