Fig. 6

Biological characterization of compound M23 in relevant RUNX1/ETO-positive -and negative cell lines. (A) Colony-forming unit (CFU) assay. Representative bar graphs of counted colonies formed by the indicated cell lines 18 days post treatment with M23, M6, 7.44, or the control DMSO at indicated concentrations. Statistics was performed using a one-way ANOVA with Dunnett’s multiple comparisons test (*p < 0.05; ****p < 0.0001 vs. DMSO-treated group). (B) Cell Titer Glo assay after 72 h was used to measure cell viability in RUNX1/ETO-positive and -negative cell lines. NC-NC cells were used as non-cancerous, human B-cell control. Curve fitting was done using a sigmoid dose-response curve and a nonlinear regression. Data was collected from three independent experiments (n = 3). (C) Cell proliferation was measured over 10 days after treatment with M23. The proliferation rates of SKNO-1 and Kasumi cells were significantly more affected than those of K562 cells. (D) Nicoletti cell cycle analysis after 24 h of treatment revealed no effect on THP-1 cells, whereas SKNO-1 cells showed an increased subG1- and G1-population and a decreased S- and G2/M- population. (E) Nicoletti assay showed that M23 induced significantly more apoptosis (% of subG1 cell population) in RUNX1/ETO-positive SKNO-1 cells than in RUNX1/ETO-negative THP-1 cells. QVD (Q-VD-OPh), a pan-caspase inhibitor, was used as a control to inhibit apoptosis induction. (F) Caspase 3/7 Glo assay after 24 h of treatment showed significantly more apoptosis induction in SKNO-1 cells in comparison to THP-1 cells. NC-NC cells served as B-cell control and no apoptosis induction was observed. (G) Differentiation analysis of SKNO-1, THP-1, and K562 cells after daily treatment with compound M23 for three days (c = 25 µM/50 µM). The percentage of CD11b-positive cells is depicted. Results are shown as means ± SD of 3 independent experiments. p values were determined by a two-tailed unpaired t-test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).