IL-34–TREM2 rapidly inactivated ERK1/2 partly via the phosphorylation of Rasal3 to induce AML cell differentiation. (A) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of C1498 cells treated with IL-34 or phosphate-buffered saline. (B) Western blot analysis of ERK1/2 protein/phosphorylation and Myc protein in MV4-11-NC and MV4-11-shTREM2 cells treated with IL-34 at the indicated concentrations (0, 50, 100, and 200 ng/mL) and time points (0, 0.5, 1, and 2 hours). (C-D) Overexpression of phosphorylated-ERK1/2 reversed the effects of IL-34 on MV4-11 cell growth (C) and colonies (D). Mean ± standard deviation (n = 3). Unpaired t test. (E-F) Overexpression of phosphorylated-ERK1/2 reversed the effects of IL-34 on C1498 cell differentiation. Scale bars, 10 μm. (G) Western blots demonstrating overexpression of phosphorylated-ERK1/2 in MV4-11 cells and resultant changes in Myc protein level in response to IL-34 treatment. (H) Scatter plot and statistics of IL-34-regulated (100 ng/mL, 30 minutes) phosphorylations in C1498 cells. (I) Coimmunoprecipitation analysis using the antibody against Rasal3 followed by western blot analysis using antibodies against phosphorylated Thr/Ser in C1498 cells treated with IL-34. (J) Western blots indicating comparable levels between phosphorylated Thr/Ser in C1498-NC and C1498-TREM2-KO cells. (K) Western blot analysis of GTP-binding Ras activity in C1498-NC and C1498-TREM2-KO cells, treated with or without IL-34. (L) Western blots demonstrating the KO of Rasal3 expression in MV4-11 cells with activated Ras GTPase in response to IL-34 treatment. Western blot images were representative of at least 3 independent experiments.