SPAG6 promoted proliferation and G1 to S phase transition in leukemic cells. (A-B,E). K562 and HEL cells were transduced with lentiviral particles expressing different shRNA clones directed against SPAG6 (shSPAG6-1 and shSPAG6-2). The cell lines HL-60 and THP-1 were transduced with lentiviral particles expressing SPAG6 or EV. The cell proliferative capacities were detected by CCK-8, colony formation, and EdU assays in K562, HEL, HL-60, and THP-1 cells with treatment as indicated. (C-D) Proliferation and colony formation of CD34⁺ cells from a patient with favorable risk (Patient 1) and a patient with poor risk (Patient 2) when SPAG6 was knocked down. (F) Western blot detection for expression levels of Ki67 and PCNA. (G) AML cell migration was determined by Transwell assay. (H) Flow cytometry analysis showing cell cycle distribution in K562, HEL, THP-1, and HL-60 cells with treatment as indicated. (I) Western blot analysis showing the expression levels of cyclin D1, cyclin E1, CDK2, and CDK4 in AML cells with treatment as indicated. Scale bar, 50 μm. *P < .05; **P < .01. PCNA, proliferating cell nuclear antigen.