Serine phosphorylation of the protein orchestrates PHF8 functions in AML. (A) Colony numbers of human THP1 AML cells transduced with ER-fused PHF8 AA (mimicking nonphosphorylated form) or PHF8 DD (mimicking phosphorylated form), allowing for an inducible expression in the presence of 4-OHT. Error bars indicate SD of 4 independent experiments. Sidak multiple comparison test (2-way ANOVA, ∗∗∗∗P < .000, ns); (lower panel) typical INT-stained colonies. (B) Number of colonies using equivalent constructs with an additional enzymatic dead F279S mutation. Error bars indicate standard deviation of 4 independent experiments (2-way ANOVA, ns); (lower panel) typical INT-stained colonies. (C) Proliferation assays, 1500 to 2000 cells were seeded in a 96-well plate and in 100 μL of medium per well. The CellTiter-Glo luminescent assay kit was used to determine the number of viable cells in culture. (D) Bar charts represent percentage of Annexin-V–positive cells analyzed by flow cytometry after 4-OHT induction. Pair t test (∗P < .05). (E) Kaplan-Meier curves for DFS of NSG mice that received transplantation via tail vein injection with 1 × 105 human THP1 cells, after 5 days antibiotic selection, transduced with indicated constructs (n = 10 mice per group). Before transplantation, mice were conditioned with sublethal radiation (2.5 Gy). The log-rank (Mantel-Cox) test was used to compare survival curves. 4-OHT, 4-hydroxytamoxifen.
