Reprogramming activities of ΔNZF-GATA-1 and ΔAD-PU.1.

(A) Expression of ΔNZF-GATA-1 in 32D/ras/ΔNZF and ΔAD-PU.1 in K562/ras/ΔAD. Total cell lysates obtained from each clone were subjected to SDS-PAGE, and the blot was probed with anti-GATA-1 antibody (left panel) or anti-PU.1 antibody (right panel). Expression levels of actin were examined as a loading control. (B) GATA-1 and PU.1 activities in 32D/ras/ΔNZF and K562/ras/ΔAD. Twenty micrograms of each reporter gene was cotransfected with 3 μg pRL-CMV-Rluc by electroporation. After 36 hours, the cells were subjected to the luciferase assays. (C) Light micrograph of 32D/ras/ΔNZF and K562/ras/ΔAD before and after 5-day culture with IPTG. Cells of each clone were cultured with or without IPTG for 5 days. Cytocentrifugation preparation from each culture was stained with May–Grünwald–Giemsa (original magnification, ×100). (D) Changes of PF4 mRNA expression before and after 5-day IPTG treatment. Total cellular RNA was extracted from 32D/ras/ΔNZF, 32D/ras/GATA-1, K562/ras/ΔAD, and K562/ras/PU.1 before and after IPTG treatment, and PF4 mRNA expression was examined by Northern blot analysis.