DAPK2 compromises erythropoiesis during anemia; exerts stage-specific proapoptotic effects; and is subject to EPO regulation in UT7epo erythroid progenitors. (A) The diagrammed construct was used to generate several independent transgenic DAPK2 founder lines. For 2 founder lines (F5 and F4), DAPK2 protein expression was assessed via Western blotting of primary splenocytes from phenylhydrazine-treated mice. (B) Erythropoiesis in Gata1-IE3.9int-DAPK2 mice is defective during phenylhydrazine induced anemia. Gata1-IE3.9int-DAPK2 mice were dosed with phenylhydrazine (52.5 mg/kg at 1 and 24 hours). Hematocrits (means ± SD) were then analyzed over a 12-day time-course. For a significant portion of Gata1-IE3.9int-DAPK2 mice, the anemia induced by phenylhydrazine was irreversible, and fatal (see Figure S5C). (C) Attenuated Epo-responsiveness in Gata1-IE3.9int-DAPK2 mice. Mice were dosed intraperitoneally with EPO (1000 U/kg). At the indicated intervals, hematocrits (means ± SD) then were determined. (D) Gata1-IE3.9int-DAPK2 erythroblast development is restricted at late developmental stages. Erythroid progenitor cells were expanded from Gata1-IE3.9int-DAPK2 and wild-type sibling bone marrow preparations. At day 3, frequencies of Kit⁺CD71^highTER119⁻, Kit⁻CD71^highTer119⁻, and Kit⁻CD71^highTer119⁺ erythroblasts were determined. For Gata1-IE3.9int-DAPK2 erythroblasts, note the decreased frequencies of Kit⁻CD71^highTer119⁺ erythroblasts. Numbers on plots are percentages of total gated cells. (E) For SP34-EX expansion cultures from Gata1-IE3.9int-DAPK2 and wild-type marrow preparations, frequencies of apoptotic cells also were assayed among developmentally staged erythroblasts (based on stage-specific cell surface markers and annexin-V staining). (F) siRNA knock-down of endogenous DAPK2 enhances UT7epo cell survival. Left panels outline the DAPK2-directed siRNA (and lentivirus vector) used, and the level of knock-down achieved (DAPK2 Western blot). The right panel illustrates increased survival of UT7epo cells due to DAPK2 siRNA. Here, cells were transduced, and isolated by FACS. Cells then were cultured in the presence of EPO (0.2 U/mL). At the hours indicated, viability was determined (via flow cytometric assay of PI positivity). Data are representative of 2 independent experiments. (G) EPO-regulation of DAPK2 kinase activity, and S318 phosphorylation. Possible effects of EPO on DAPK2 activity were studied in UT7epo cells stably transduced (at low MOI, and FACS-isolated) with a CGW-(flag)DAPK2 lentivirus. Here, UT7epo-(flag)DAPK2 cells were cultured for 16 hours in the absence of hematopoietic cytokines, and then exposed to EPO (5 U/mL) for the indicated intervals. The activity of immunoprecipitated (flag)DAPK2 then was assayed in vitro using MLC as a substrate (top panel). Assays of P-Ser19-MLC were by Western blotting (myosin light chain-2 p-Ser19 antibody; Cell Signaling Technology, Danvers, MA). Effects on DAPK2 phosphorylation at an inhibitory Ser318 site also were analyzed (bottom panel; DAPK2 p-Ser318 antibody; Santa Cruz Biotechnology, Santa Cruz, CA).