Figure 1

Optimization of AzaC treatment protocol. (a) AML3 cells were treated three times with vehicle, 0.5, 1, or 2 μM AzaC (triangle) at 0, 24, 48 h, harvested at 96 h, and western blotted for DNMT1. (b) AML3 cells were treated with vehicle, 0.5, 1.5, or 5 μM AzaC at 12-h intervals and viability determined by fluorescence assay (Resazurin) at indicated time points. (c) AML3 cells were treated three times with vehicle, 0.5, 1, or 2 μM AzaC (triangle) at 0, 24, 48 h and whole cell lysates western blotted for γH2AX at 72 h. As a positive control, cells were treated with vehicle or 1 μM etoposide (Eto). (d) AML3 cells were treated three times with vehicle, 0.5, 1, or 2 μM AzaC (triangle) at 0, 24, 48 h, harvested at 72 h, and western blotted for uncleaved and cleaved PARP. As a positive control, p53 inducible SAOS2 cells were treated with vehicle or doxycycline (Dox). (e) AML3 cells were treated three times with vehicle, 0.5 or 5 μM AzaC (as indicated by triangle) at 0, 24, 48 h and cumulative cell divisions scored by CFSE at 96 h. Results from three replicate experiments with SD. (f) AML3 cells were treated three times with vehicle, 0.5, 1, or 2 μM AzaC (triangle) at 0, 24, 48 h, pulse labelled with BrdU for 1 h at 96-h time point, and DNA content (7-AAD) and DNA synthesis (BrdU) determined by FACS. (g) AML3 cells were treated three times at 24-h intervals with 0.5 μM AzaC in triplicate and harvested 96 h after the first treatment. Number of CpGs showing decreased (hypo) and increased (hyper) methylation in AzaC treated cells, compared to untreated cells. (h) Overall percent methylated basecalls for all reference CpGs, in cells from (g).