Dominant-negative forms of STAT5A inhibit Epo-induced SKT6 cell hemoglobinization and globin expression. SKT6 cells were transfected stably with expression vectors encoding forms of STAT5A in which either the predicted DNA binding domains (pCIneo-S5▵D, pMK-S5▵D) or the C-terminal transactivation domains were deleted (pMK-S5▵C) (Wang-Ihle). As a control, a STAT3 DNA binding domain deletion mutant (S3▵D) also was constructed and was expressed stably (SKT6-pCIneo-S3▵D cells). Effects of the expression of these STAT mutants on Epo-induced SKT6 cell differentiation were then assessed. (A) Shown diagrammatically are wt STAT5A, wt STAT3, and the derived deletion constructs S5▵D, S5▵C, and S3▵D. (B) Levels of expression of STAT5A deletion constructs in SKT6-pCIneo-S5▵D, -pMK-S5▵D, and -pMK-S5▵C cells as assayed by Western blotting of total cell lysates. The designated antibody S5#1 recognizes a C-terminal epitope of murine STAT5A (Asp⁷⁷⁴-Ser⁷⁹³), whereas S5#2 recognizes an epitope within a central domain (Phe⁴⁵¹-Tyr⁶⁴⁹) of STAT5-A and -B. As controls, lysates from parental SKT6 cells as well as STAT5A immunoprecipitated from SKT6 cells were coanalyzed. (C) In assays of induced hemoglobinization, SKT6 cells expressing either STAT5▵D, STAT5▵C, or STAT3▵D (as a control) were exposed to Epo (2 U/mL) and, at the indicated intervals (48 and 72 hours), hemoglobinized cells were stained with DAF and scored (>200 cells per sample). Values are the mean frequencies of DAF-positive cells ± standard deviations (n = 3). Frequencies of DAF staining-positive cells scored in the absence of Epo were subtracted as background from these numbers. (D) In assays of induced globin expression, SKT6-pCIneo-S5▵D and SKT6-pCIneo-S3▵D cells (top panel) or SKT6-pMK-S5▵C and SKT6-pMK-S5▵D cells (lower panel) were exposed to Epo (2 U/mL) and, at 72 hours, cell lysates were prepared. Globin levels then were assayed by Western blotting. As a positive control, lysates from parental SKT6 cells (±Epo exposure) were coanalyzed.