Fig. 5
Clonal heterogeneity in SAMHD1 levels drives intrinsic resistance to CNDAC but not population doubling time in MV4-11 cells. (A) Schematic illustration of the establishment of MV4-11 single cell-derived clones by limiting dilution. (B) CNDAC concentrations that reduce viability of 12 single-cell-derived MV4-11 clones by 50% (IC50). Values represent means ± SD of three independent experiments. (C) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMHD1), and DCK in single cellderived MV4-11 clones. GAPDH served as a loading control. (D) Analysis of SAMHD1 promoter methylation in MV4-11 clones through amplification of a single PCR product (993-bp) corresponding to the promoter sequence after HpaII digestion. (E) Correlation of the CNDAC IC50 values with cellular SAMHD1 or DCK protein levels, quantified using nearinfrared Western blot images to determine the ratio SAMHD1/ GAPDH or DCK/ GAPDH. Closed circles and error bars represent means ± SD of three independent experiments, each performed in three technical replicates. Linear regression analyses were performed using GraphPad Prism. (F) Western Blots and IC50 values for CNDAC and daunorubicin in MV4-11 clones 9, 11, and 12 after transfection with SAMHD1-siRNAs (siSAMHD1) or non-targeting control siRNAs (siCTRL). Each symbol represents the mean ± SD of three technical replicates of one representative experiment out of three. P-values were determined by two-tailed Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001). (G) Population doubling time (PDT) in MV4-11 single cell-derived clones and correlation of the PDT with cellular SAMHD1 or DCK protein levels. Closed circles and error bars represent means ± SD from the quantification of three Western Blots. Linear regression analyses were performed using GraphPad Prism