PML4 promotes terminal erythroid differentiation through GATA-1. (A-C) Forced expression of PML4 promotes K562 erythroid differentiation. K562 cells were infected with a retrovirus that expressed PML4 cDNA. (A) Western blot was performed to detect overexpression of PML. (B) Real-time RT-PCR was performed for AHSP and globin gene expression. (C) Flow cytometry was used to detect expression of GPA. (D-F) PML knockdown in K562 cells leads to reduced expression of erythroid genes. K562 cells were infected with a retrovirus harboring an shRNA to knock down PML (or GFP as a control). (D) Western blot assay, (E) real-time RT-PCR, and (F) flow cytometry were performed as in A-C. (G) Human primary erythroid progenitors were expanded and induced to erythroid differentiation by EPO. At day 1 of differentiation, retroviral vectors for PML4 overexpression or PML knockdown were transfected. PML and β-like globin genes expression were determined on day 4 of differentiation. (H-J) PML4 promoted erythroid maturation of G1E-ER4 but not G1E. PML4 and control plasmids were stably overexpressed in G1E and G1E-ER4 cells by retrovirus infection and induced to erythroid differentiation by adding β-estradiol for 18 hours of treatment. (H) Western blotting analysis of GATA-1 and PML4 expression in PML4 overexpressing and control G1E, G1E-ER4 cells before and after β-estradiol induction. (I) Real-time RT-PCR analysis of AHSP and α- and β-globin genes expression in PML4 overexpressing and control G1E and G1E-ER4 cells before and after β-estradiol induction. Right panel shows the enlarged bars from G1E cells. (J) Benzidine staining of PML4 overexpressing and control G1E-ER4 cells before and after 18 hours of β-estradiol induction for GATA-1 recovery. PML4 promotes hemoglobin production, especially in β-estradiol–induced G1E-ER4 cells.