Functional implication of HDAC5 as a target of erythropoietin signaling. (A-B) HDAC inhibition partially substitutes for erythropoietin signaling during GATA1–induced erythroid differentiation. G1ER cells induced with estradiol ± erythropoietin (Epo) and ± SAHA were assessed for differentiation in panel A and viability in panel B. Data are mean ± SEM for 3 experiments. (C-E) G1ER cells transduced with shRNA constructs targeting HDAC5 were assessed for HDAC5 and HDAC4 expression, erythroid differentiation, and viability. (C) Immunoblot (IB) of whole cell lysates. sh 253 provided strong knockdown of HDAC5, and sh 252 provided no knockdown of HDAC5. (D) Differentiation of cells induced with estradiol and erythropoietin (0.5, 0.1, or 0.05 U/mL). *P = .01 for sh 253 vs control at corresponding Epo dose in 3 experiments. (E) Viability of cells induced with estradiol and erythropoietin. *P = .03. See also supplemental Figure 3 for HDAC4 knockdown data. (F-G) Distinct subcellular localization patterns of HDAC5 and HDAC4 in erythroid progenitors. (F) G1ER cells stimulated with erythropoietin (Epo) for the indicated durations were subjected to subcellular fractionation followed by immunoblotting for HDACs. Poly(ADP-ribose) polymerase and lactate dehydrogenase served as loading controls for nuclear and cytosolic fractions, respectively. (G) Human CD34⁺ progenitors grown in erythroid medium for the indicated durations were analyzed as in panel F.