Activation of the HOXB4 promoter by TAT-NF-Ya. (A) K562 cells were electroporated with a HOXB4 promoter-luciferase construct together with the plasmids that encode NF-Ya partners: NF-Yb, NF-Yc, and USF1, USF2 (gray bars). K562 subline stably transformed with HOXB4-luciferase was also used (black bars). Transiently and stably transfected K562 cells were incubated with TAT-NF-Ya for 90 minutes and then analyzed for firefly luciferase activity. Activity was normalized to Renilla luciferase and then compared with the luciferase activity of control (transfected K562 cells without TAT-NF-Ya treatment). (B) Endogenous HOXB4 mRNA expression measured by real-time PCR in K562 unmanipulated cells (1), treated with TAT-β-Gal (2), or treated with TAT-NF-Ya (3). (C) Time course of HOXB4 mRNA induction after treatment with TAT-NF-Ya. Human PB CD34⁺ cells were treated with 60nM TAT-NF-Ya plus 250mM sucrose, then cultured with myeloid cytokines (“TAT-NF-Ya protein delivery to the cells”) from 2 to 15 days, and endogenous HOXB4 mRNA was measured by quantitative PCR. Compared with CD34⁺ cells cultured in cytokines without TAT-NF-Ya, HOXB4 mRNA increased 2.5- to 3-fold from day 2 to day 7 and remained elevated throughout the culture.