Figure 1

FICZ augments RA-induced differentiation. HL-60 cells were untreated (C, control) or treated with FICZ, RA, or RA plus FICZ for indicated times. FICZ augments RA-induced expression of differentiation markers. A. CD38 expression (assessed by flow cytometry with APC-conjugated antibody) at 6 h post treatment is not significantly modulated either by FICZ alone or in combination with RA. B. CD11b expression (assessed by flow cytometry with APC-conjugated antibody) was increased by the combination therapy compared to RA alone after 24 h (p = 0.012), C. 48 h (p = 0.042) and D. 72 h (p = 0.0029) of treatment. Flow cytometric assay of live cells was carried out setting the logical gate to exclude 95% of the untreated cells during CD38 and CD11b detection. E. Superoxide metabolites from inducible oxidative metabolism were measured using flow cytometry of DCF stained cells. Respiratory burst is not significantly enhanced at 48 h by combination therapy compared to RA alone (p = 0.08). F. Respiratory burst, measured by flow cytometry of DCF stained cells, is significantly enhanced at 72 h by co-treatment compared to RA alone (p = 0.001). G. Cell density was measured for control and treated cells at progressive times, 0, 24, 48 and 72 h. Cell growth shows that FICZ at the concentration used has no toxic effect. FICZ by itself does not decrease the cell number, however it enhances RA-induced growth inhibition. H. Cell cycle distribution was measured by flow cytometry of propidium iodide stained nuclei. The percent of cells in G0/G1 is shown. G0/G1 cell-cycle arrest was propelled by co-treatment compared with RA alone both at 48 h (p = 0.009) and I. 72 h(p = 0.035) as shown by flow cytometry of DNA-stained nuclei.