Fig. 3: Dactinomycin induces nucleolar stress in NPM1-mutated AML cells in vitro and in patients.

A Immunofluorescence of OCI-AML3 cells treated with dactinomycin at the indicated concentrations for 1 h/day for a maximum of 4 consecutive days, stained with an anti-NPM1, which recognizes both mutant and wild-type NPM1 (tNPM1). B Western blot analysis of TP53 and β-actin in OCI-AML3 cells treated with dactinomycin at the indicated concentration for 1 h. C Viability of OCI-AML3 cells treated with dactinomycin at the indicated concentration for 1 h/day for 4 consecutive days and studied by flow cytometry with 7-AAD and Annexin V. Results are expressed as percentage of 7-AAD/Annexin V double-negative cells (n = 4, Dunnett’s multiple comparison). D Immunofluorescence of PDX2 cells treated with dactinomycin at the indicated concentrations for 1 h/day for a maximum of 4 consecutive days, stained with the anti-tNPM1. E Western blot analysis of TP53 in PDX2 cells treated with dactinomycin at the indicated concentration for 1 h. F Viability of PDX2 cells treated with dactinomycin at the indicated concentration for 1 h/day for 4 consecutive days and studied by flow cytometry with 7-AAD and Annexin V. Results are expressed as percentage of 7-AAD/Annexin V double-negative cells (n = 4, Dunnett’s multiple comparison). G Immunofluorescence of cells isolated from the bone marrow of patient UPN002 before starting the treatment (Pre), 4 h following the first administration (d1), and 4 h after the fourth administration (d4). Cells were stained with the anti-tNPM1. H Western blot analysis of TP53 in cells isolated from the bone marrow of patient UPN002 before starting the treatment (Pre), 4 h following the first administration (d1), and 4 h after the fourth administration (d4). ActD, dactinomycin. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate s.e.m. Scale bar: 10 μm.