ZDHHC21 activates OXPHOS to arrest the myeloid differentiation of AML cells. (A) Gene set enrichment analysis plots depicting enrichment of HSC up gene signature and myeloid cell development down gene signature in the transcriptional profiling of samples with high ZDHHC21 expression in TARGET database. (B) RNA sequencing analysis of HL60 cells infected with lentivirus-shZDHHC21 (#1 and #2) for 4 and 7 days. (Top) Schematic representation of comparing gene expression profiles in HL60 cells. Overlapped smaller circles reflected the shared genes induced by different lentivirus-shZDHHC21s on day 4 (left; blue) and day 7 (right; purple), respectively. Overlapped smaller oval reflect the shared 693 genes induced on both day 4 and day 7. Red numbers in circles represent upregulation, whereas blue numbers represent downregulation. (Bottom) The functional clustering analysis of the changed genes after overlapping screening using Metascape. The −log₁₀(P) values mean the enrichment score, representing the degree of participation. The number of enriched genes is also shown. (C) The expression levels of ZDHHC21 in primary AML cells or normal hematopoietic cells of various lineages were obtained from the BloodSpot database. (D) RNA sequencing analysis showed the messenger RNA changes of differentiation marker genes (C/EBPB, CD18, IL1B, CD11C, CD18, CD14, MAFB, CSF1R, and CD11B) in HL60 cells infected with lentivirus-shZDHHC21. (E) CD11b expression of HL60 cells infected with lentivirus-shZDHHC21 or lentivirus-shZDHHC23 for 7 days. (F) CD11b expression of different AML cell lines infected with lentivirus-shZDHHC21. (G) CD14 expression of HL60 cells infected with lentivirus-shZDHHC21 for 7 days. (H) Cell differentiation of HL60 cells infected with lentivirus-shZDHHC21 or lentivirus-shZDHHC23 for 7 days. (Top row) Cell morphological analysis after Wright-Giemsa staining. (Bottom row) NBT-reducing activity. Data are representative of at least 3 individual experiments, and 1 representative image is shown. (I) Quantitative analysis of NBT-reducing activity of HL60 cells infected with lentivirus-shZDHHC21 or lentivirus-shZDHHC23 for 7 days. (J) Bacterial killing ability of HL60 infected with shZDHHC21 lentivirus for 7 days. Clearance efficiency was determined from the numbers of viable bacteria recovered from the intracellular compartment after infection. Staphylococcus aureus was used to infect AML cells. (K) The cell proliferation assay showed proliferation inhibition caused by ZDHHC21 knockdown in HL60 cells. (L) Flow cytometry analysis of cell cycle distribution in HL60 cells after silencing ZDHHC21 for 5 days. Data are presented as mean ± SD (n = 3). n.s., P > .05; ∗P < .05; ∗∗∗P < .001 vs shCtrl. The significance analysis was conducted by a two-tailed unpaired t test or one-way analysis of variance. BC, band cell; CMP, common myeloid progenitor; early_PM, early promyelocyte; FDR, false discovery rate; GMP, granulocyte-monocyte progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte-erythrocyte progenitor; late_PM, late promyelocyte; MM, metamyelocyte; mono, monocyte; MPP, multipotent progenitor; MY, myelocyte; NES, normalized enrichment score; PMN, polymorphonuclear cell.