Fig. 4
HMA resistance development in HL-60 cells results in SAMHD1 upregulation. a Representative immunoblots of SAMHD1, other proteins involved in AZA and DAC metabolism, and DNA methylation (DNMT1) in parental HL-60 cells and in three AZA- or DAC-resistant HL-60 sublines (rAZA I–III, rDAC I–III). β-Actin served as a loading control. b, c IC50 values for (b) DAC and (c) AZA in AZA- and DAC-resistant cell lines relative to the parental counterpart. Each circle represents a technical replicate (n = 3) of three independent experiments performed. Horizontal lines and error bars represent means ± s.d. The numbers above the data points indicate the factor of increase of IC50 values in resistant cells relative to parental HL-60 cells. d Parental, AZA- and DAC-resistant HL-60 cells were treated with the VLPs indicated and subsequently analyzed for AZA and DAC cytotoxicity (top) and SAMHD1 expression (bottom). IC50 values of three independent experiments each performed with technical replicates (n = 3) are presented as closed circles. Horizontal lines and error bars represent means ± s.d. Numbers above the bars indicate the factor of decrease in IC50 values in Vpx-VLP-treated cells relative to Vpr-VLP-treated controls. e, f Representative LC–MS/MS analyses of (e) DAC-TP and (f) AZA-TP in parental and AZA- or DAC-resistant HL-60 cells, treated with Vpx-VLPs (yellow/light blue chromatogram), with control Vpr-VLPs (dashed orange and dark blue chromatogram) or left untreated (orange and dark blue chromatogram). g Analysis of SAMHD1 promotor methylation through amplification of a single PCR product (993-bp) corresponding to the promoter sequence after HpaII digestion. A 0.25-kb fragment of the GAPDH gene lacking HpaII sites was PCR-amplified using the same template DNA served as loading control, THP-1 and Jurkat cells served as positive and negative controls, respectively. h Global DNA methylation determined by measurement methylation levels of long interspersed element (LINE)-1 in parental HL-60 cells and rAZA or rDAC HL-60 sublines. Each circle represents means ± s.d. of a technical replicate (n = 3) of one representative experiments out of three
