Fig. 4: Mechanistic evaluation of 20D9h3-DUBA and 20D9h3-MMAF.

A MV4-11 cells were treated with 100 ng/ml 20D9h3-DUBA (left) or 20D9h3-MMAF (right) for 24-96 h. Annexin V-positive and DAPI-positive cells were assessed every 24 h by flow cytometry and categorized as live cells (double negative), early apoptotic (Annexin V-positive), late apoptotic (double positive) or dead cells (DAPI-positive). mean±s.d.; n = 4 biological replicates. B MV4-11 cells were treated with 100 ng/ml 20D9h3-DUBA or 20D9h3-MMAF for 48 h. Cells were fixed with 80% (v/v) EtOH and stained with PI to evaluate cell cycle phase. The percentage of cells in G1, S and G2 was calculated with FlowJo version 10.8.1 using the Watson pragmatic algorithm. Values are depicted as ratio to untreated control. mean±s.d.; n = 4 biological replicates. **P < 0.01; ****P < 0.0001; ns, not significant by One-way ANOVA; each p value is adjusted to account for multiple comparisons. C MOLM-13 cells were treated with 50 ng/ml 20D9h3-DUBA or 20D9h3-MMAF or left untreated for 24 h. Afterwards, ATR, p-ATR, CHK1 and p-CHK1 expression was assessed by Western blotting using beta-Actin as loading control. Blots are representative of three independent experiments. MW = molecular weight. D FLT3-positive MOLM-13 cells stably expressing luciferase (luc) and mCherry and FLT3-negative native HL-60 cells were co-cultured at a ratio of 8:1 for 5 d in the presence of different doses of 20D9h3-DUBA or 20D9h3-MMAF. Viable cells were counted by flow cytometry using LIVE/DEAD™ Fixable Aqua Dead Cell Stain and normalized to untreated control. The two cell types were distinguished by mCherry signal. mean±s.d.; n = 3 biological replicates. E MOLM-13 luc mCherry cells with empty vector (ev) or ABCB1 expression were treated with a dilution row of 20D9h3-DUBA, 20D9h3-MMAF or GO for 96 h. Viable cells were assessed with resazurin readout and were normalized to untreated control. mean ± s.d.; n = 3 biological replicates.