Silencing KDM5B inhibited proliferation and induced apoptosis in primary AML cells and certain human AML cell lines. (A–C) The primary human AML cells were collected and transduced with lentivirus expressing scramble (shScr) and shRNAs. The mRNA level of KDM5B was measured by RT-qPCR and normalized by Actb (A). Proliferation was measured by Cell Counting Kit-8 (CCK-8) assay (450 nm) (B). The cell apoptosis was measured by Annexin V staining and analyzed/visualized by flow cytometry and Flowjo (C, right) Quantification from triplicates. The cells were collected from three AML patients (n = 3). The clinical information for these samples is provided in Supplemental Table S1. (D–H) MOLM-13 cells were transduced with lentivirus expressing scramble (shScr) and shRNAs. The relative mRNA and protein levels of KDM5B were measured by RT-qPCR (D) and western blotting (E), respectively. Cell apoptosis (F) was measured by Annexin V staining, and proliferation was measured by respective CCK-8 assay (G) and BrdU staining (H). (I) The colony-formation ability of KDM5B KD and control MOLM-13 cells was measured in a methylcellulose medium (STEMCELL Technologies, Cat #: M3434). (J) MOLM-13 cells were infected with lentivirus expressing GFP control and GFP-KDM5B, and the percentage of GFP+ cells was measured at indicated times. The percentage of GFP-positive cells was measured by flow cytometry, which was further analyzed by Flowjo software. (D–J) Quantification from three biological repeats. (K,L) After exposure to a nonlethal radiation dose (1 Gy), 2 × 106 MOLM-13 cells with control and KDM5B KD were transplanted into NOD mice. The spleen size/weight (K, left) and leukocyte number (K, right) were measured and recorded 2 wk after transplantation. The survival of the remaining mice was monitored (L). n = 5 for each comparison. Data represent the mean + SD (A, C, D–F, and H–K) or ±SD (B,G). (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test (A–K) and log-rank test (L).
