FIG. 6.

HERP2 inhibits transcriptional activation by GATA-1 and erythroid differentiation of K562 cells, and HERP2 siRNA interferes with c-Jun-mediated inhibition and augments GATA-1 function. (A) Analysis of transcriptional activation by GATA-1 with and without either HERP2 or EPAS-1. Reporter assays were carried out as described for Fig. 2. (B) K562 cells transduced with the indicated retroviral constructs were subjected to erythroid induction followed by benzidine staining for hemoglobin. (C) Analysis of transcriptional activation by GATA-1 with and without siRNA for HERP2 and with and without c-Jun; 2 × 10⁶ K562 cells were transfected with 2 μg of pEF-GATA-1, 3 μg of pSUPER or pSUPER-siHERP2.2, 1 μg of pCMV-c-Jun, 1.5 μg of αIIb-598-Luc, and 0.5 μg of pCMV-βgal, as indicated. After 72 h of incubation, cells were harvested for standard luciferase and β-galactosidase assays. Results shown represent three independent experiments ± standard error of the mean. (D) Demonstration of Flag-HERP2 knockdown by the HERP2 siRNA construct. K562 cells cotransfected with pcDNA3.1(−)-Flag-HERP2 plus either the pSUPER vector or pSUPER-siHERP2.2 were analyzed by immunoblotting (IB) for Flag or tubulin. Transfections were carried out with 5 μg of DNA per 2 × 10⁶ cells. Ratios below the panels indicate the relative amounts (in micrograms) of the Flag-HERP2 and siRNA vectors, respectively.