PUM1 and PUM2 contribute to human leukemic cell growth. (A) RT-qPCR analysis of hPUM1 and hPUM2 mRNA expression in AML patient samples grouped according to the French-American-British classification, and in CD34+ cells from healthy donors (peripheral blood or cord blood or BM). Each symbol represents data from a single patient. CD34+: 11 samples; AML0: 5 samples; AML1: 16 samples; AML2: 26 samples; AML3: 7 samples; AML4: 6 samples; AML5: 4 samples. Results are normalized to GAPDH, HPRT, and Cyclophilin A transcript levels. (B-E) MOLM-14 leukemia cells were transduced or not (nt) with the indicated shRNA-encoding vectors. (B) Immunoblot analysis of the indicated proteins at day 3 posttransduction. Actin was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 3). (D) Cell cycle analysis upon propidium iodide labeling at day 3 posttransduction (n = 3). (E) Annexin V labeling at day 4 posttransduction (n = 3). (F) Primary AML patient samples were transduced with shCtrl-, shPUM1-, or shPUM2-encoding vectors, and cultured onto MS5 stromal cells in CD34+ growth medium. At day 10, the cultures were disrupted by vigorous pipetting and scraping, and nucleated cells were counted by FACS. Results are expressed relative to shCtrl-transduced cell numbering (n = 6). Each symbol represents data from a single patient. Data are expressed as mean ± SEM. Ns, not significant. *P < .05; **P < .01; ***P < .001 (Mann-Whitney test in panel A; Student t test in panels C-F).
