Fig. 1

SAMHD1 (but not DCK) levels determine sensitivity to CNDAC and inversely correlate with CNDAC-triphosphate (CNDAC-TP) in leukaemia cell lines. (A) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMHD1), and DCK in 13 AML cell lines. GAPDH served as loading control. Uncropped Western blots are presented in Supplementary Figure 1. (B) CNDAC concentrations that reduce the viability of AML cell lines by 50% (IC₅₀). Horizontal lines and error bars represent means ± SD of three independent experiments. (C) CNDAC triphosphate (CNDAC-TP) levels determined by LC–MS/MS. Horizontal lines and error bars show means ± SD of three independent experiments. (D, E) Correlation of the CNDAC IC50 values with cellular DCK (D) or SAMHD1 (E) protein levels, quantified using near-infrared Western blot images to determine the ratio DCK/ GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (F, G) Correlation of CNDAC-TP levels with cellular DCK (F) or SAMHD1 (G) protein levels in AML cell lines, quantified using near-infrared Western blot images to determine the ratio DCK/GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (H) Analysis of SAMHD1 promoter methylation in AML cell lines 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 served as control cell for an unmethylated SAMHD1 promotor, while JURKAT served as control cell for a methylated promotor. (I) Correlation of CNDAC IC₅₀ values in 26 ALL cell lines (11 T-ALL, 15 B-ALL) with SAMHD1 protein levels, quantified using near-infrared Western blot images to determine the ratio SAMHD1/ GAPDH relative to the positive control THP-1. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (J-L) Comparison of SAMHD1 protein levels (J), CNDAC IC₅₀ values (K) and CNDAC-TP levels determined by LC-MS/MS (L) in T-ALL and B-ALL cells. Each point represents the mean of three independent experiments. One-tailed Student’s t-tests were used to compare means in T-ALL and B-ALL cells (represented as horizontal lines ± SEM)