OTKs enhance the expression of POLθ. (A-C) Western analysis of POLθ expression in nuclear lysates from Lin^-CD34⁺ cells from healthy donors (normal) and patients with FLT3(ITD)-positive AML, Jak2(V617F)-positive MPN (PV, ET, and PMF), and BCR-ABL1–positive CML (CP and BP) (A) and parental (32Dcl3, BaF3) cells and these expressing the indicated oncogenes (B). Lamin served as loading control. (C) Western blot of indicated proteins in total cell lysates from cells expressing OTKs and treated with specific TKi (FLT3 inhibitor AC220, JAK1/2 inhibitor ruxolitinib, and ABL1 inhibitor imatinib). (D) CHX chase assay: nuclear cell lysates obtained from indicated cells after CHX treatment (hours) were analyzed using western blot to detect POLθ and lamin (loading control). (E) Western blot of indicated proteins in total cell lysates from cells expressing OTKs and treated with specific TKi (FLT3 inhibitor AC220, JAK1/2 inhibitor ruxolitinib, ABL1 inhibitor imatinib) and proteasome inhibitor MG132. BP, blast phase; CHX, cycloheximide; CP, chronic blast; ET, essential thrombocythemia; PMF, primary myelofibrosis; PV, polycythemia vera.