Lineage- and age-dependent effects of Trsp KO hematopoiesis. (A) Schematic representation of the pathways involved in Sec incorporation into selenoproteins. This process involves a multiprotein complex that includes tRNA Sec 1 associated protein 1 (TRNAU1AP), eukaryotic elongation factor (EEFSEC), and tRNA^Sec isoform by SECIS binding protein 2 (SECISBP2). SECISBP2 interacts with the SECIS stem-loop element located in the 3′ untranslated region of mammalian selenoprotein mRNAs, enabling the decoding UGA Sec codons at the ribosomal acceptor site to mediate Sec incorporation into nascent polypeptide. (B) Utilization of a mouse model allowing time-dependent deletion of the tRNA^sec gene (Trsp) through the Cre-loxP system in Trsp^fl/fl: Mx1-Cre mice. Trsp gene is flanked by loxP sequences, shown by arrowheads. Trsp excision was induced by intraperitoneal (IP) injection of pI-pC every other day for 3 times. Control mice were pI-pC–treated Trsp^fl/fl mice, whereas Trsp KO mice represent Trsp^fl/fl: Mx1-Cre mice. (C) Western blot analysis for SEPHS2, GPX1/2, and GPX4 in whole BM cells and spleen cells derived from control mice and Trsp KO mice. Filled triangles indicate target protein bands, whereas open triangles represent nonspecific bands. (D) Absolute number and frequency of CD71⁺Ter-119⁺, CD3⁺, B220⁺, CD11b⁺Gr-1⁺, and CD11b⁺Gr-1^– cells in the BM are shown. Frequencies of CD71⁺Ter-119⁺, CD3⁺, B220⁺, CD11b⁺Gr-1⁺, CD11b⁺Gr-1^– cells, PB, and spleen of each group are shown (n = 5 per group). (E) Frequency of B220⁺ and CD11b⁺Gr-1⁺ cells in the BM of each group (young represents age <20 weeks; aged, 81 weeks; n = 5 per each group). (F) Representative histograms and mean fluorescence intensity (MFI) of C11-BODIPY 581/591 and CellROX staining in B220⁺ or CD11b⁺ BM cells of aged control and Trsp KO mice (n = 5 per group; age 81 weeks). (G) Schematic representation of noncompetitive BM transplantation assays. (H) Counts of white blood cells (WBCs) in whole blood from recipient mice at 3 months after transplantation (control group, n = 6 mice; Trsp KO group, n = 8 mice). (I) Frequency of CD45.2⁺ in the PB and of B220⁺ and CD11b⁺Gr-1⁺ cells in CD45.2⁺ cells of recipient mice at 3 months after transplantation (control group, n = 6 mice; Trsp KO group, n = 8 mice). P values were calculated by a 2-sided Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.