Additional file 1: Table S1. of 5’UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia

Main clinical and laboratory features of the AML patients with ANKRD26 mutations identified by the present investigation. Table S2. Prediction of translation start codon presence in the ANKRD26 coding sequence and relative protein size. Figure S1. The stability of ANKRD26 mutant proteins is similar to that of the WT counterpart. HeLa cells were transfected by ANKRD26-FLAG WT or mutant constructs and cultured in a 12-well plate. Protein synthesis was blocked 24 h after transfection by addition to the cell culture of cycloheximide 100 mM diluted in DMSO. Control conditions were carried out by adding the same amount of DMSO alone. Cells were then lysed just before the addition of cycloheximide or DMSO (time 0) and 8, 24, and 48 h after the addition of cycloheximide or DMSO and analyzed by immunoblotting with anti-FLAG and anti-tubulin antibodies. The histograms show the amount of proteins expressed as FLAG/tubulin ratio and referred to time 0 of each condition. After the addition of cycloheximide, WT ANKRD26 expression decreased to about 60% at 8 h, to 45% at 24 h, and to 20% at 48 h. The expression was significantly lower after cycloheximide treatment compared with DMSO alone at each time point, indicating that protein synthesis was efficiently blocked by cycloheximide (***P < 0.001; **P < 0.01; *P < 0.05). Overall, mutant and WT proteins showed a similar kinetic of reduction after cycloheximide treatment. Data reported represent the mean of three independent experiments and are reported as mean ± S.E.M. Statistical analysis was performed by Student t test. Methods. (DOCX 200 kb)