ZDHHC6-mediated palmitoylation restrains FLT3-ITD surface expression, signaling and colonogenic growth in primary human AMLs. (A) Examination of FLT3 palmitoylation in primary FLT3-WT and FLT3-ITD⁺ AML cells by the APE assay. (B) Flow cytometric plots showing cell surface FLT3 expression of various FLT3-WT (black traces) vs FLT3-ITD⁺ (red traces) primary AMLs. Control (shaded area) indicates secondary antibody only. (C) The quantification of the MFIs of surface FLT3 levels in FLT3-ITD⁺ AMLs (left; n = 5) relative to that in FLT3-WT AMLs (n = 6), as shown in panel B. Relative surface/total FLT3 level in FLT3-ITD⁺ AMLs (right) when compared with FLT3-WT AMLs. The total FLT3 levels were determined by flow cytometry after fixation and permeabilization of AML cells. (D) Quantification of the MFIs of surface FLT3 level (left) and relative surface-to-total FLT3 level (right) in FLT3-ITD⁺ AML cells depleted of ZDHHC2 or ZDHHC6 when compared with that of Luc control (n = 3 for each group). (E) WB analysis of signaling transduction in 2 FLT3-ITD⁺ patient AML cells depleted of ZDHHC2 or ZDHHC6 vs Luc. (F) Cell growth of FLT3-ITD⁺ patient AML cells upon depletion of ZDHHC2 or ZDHHC6 as in panel D. (G) Relative colony-forming capacity of 3 individual FLT3-ITD⁺ patient AML cells with depletion of ZDHHC2 or ZDHHC6 vs Luc. All relevant data are presented as means ± standard deviation. (C-D) Each symbol represents individual patient AML cells. (F-G) Data analyses were performed in triplicate. In all relevant panels, *P < .05; **P < .01; ns, not significant, as determined by 2-tailed Student t test.