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K.N. Smitheman et al.
tant decrease in S phase (Online Supplementary Figure S1C). No appreciable accumulation of cells in sub-G1 was observed. To confirm the cell cycle findings, a BrdU assay that measures actively dividing cells by incorporation of BrdU into DNA during S-phase was performed (Figure 1B). MOLM-13 cells treated for six days had a dose
responsive decrease in BrdU signal (EC50 = 1.9±0.9 nM). Finally, induction of caspase 3/7 was assessed to deter- mine if cells were activating the apoptosis pathway. Seven AML cell lines were treated for up to six days with GSK2879552. In six of seven AML cell lines there was no appreciable induction of caspase 3/7 at any time point
C
A
B
Figure 5. The combination of GSK2879552 and all-trans retinoic acid (ATRA) promotes caspase-mediated cell death. (A) GDI values (±Standard Error) of MOLM- 13 (left) and OCI-AML3 (right) cells treated with 1000 nM GSK2879552, 100 nM ATRA, or 1000 nM GSK2879552 + 100 nM ATRA (COMBO) for 1 to 6 days. †Significance (P<0.05) between GSK552 and COMBO. *Significance (P<0.05) between GSK552 and COMBO as well as between ATRA and combination (COMBO). (B) Fold caspase 3/7 cleavage values from day of peak induction (blue bars, ±Standard Error) for MOLM-13 cells (top) and OCI-AML3 cells (bottom) treated with the indicated concentrations of GSK2879552 and ATRA. The value representing the additive effect at each combination concentration is indicated with a red circle. (C) Shading represents fold caspase 3/7 cleavage values relative to threshold from day of peak induction of AML cells treated with the indicated concentrations of GSK2879552 and ATRA. Dark green shading indicates >10-fold above additive threshold, light green indicates 5- to 10-fold above additive threshold, light brown indicates 1- to 5-fold above additive threshold.
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haematologica | 2019; 104(6)