Gfi1 mRNA levels decrease, Gfi1 protein levels increase during monocytic differentiation. (A) Gfi1 quantitative RT-PCR was performed on RNA samples of U937 and HL60 cells during PMA- or ATRA-induced differentiation (n = 3). Gfi1 expression levels in untreated HL60 and U937 cells were set at 100% and values were normalized for β-actin expression. Error bars indicate standard deviation. (B) Cell lysates of PMA-differentiated U937 cells taken at indicated time points were immunoblotted and stained with an α-Gfi1 antibody (N20; Santa Cruz Biotechnology). Lamin staining shows equal loading. (C) U937 cells were incubated with PMA. After the indicated pulse times cells were washed and harvested 72 hours after the beginning of the experiment. Numbers below the blot indicate the x-mean of CD11c expression. (D) Gfi1 levels in HL60 cell lysates taken after 24-, 48-, and 72-hour treatments with ATRA or PMA were compared with untreated cells showing that Gfi1 is strongly induced upon monocytic differentiation. Lamin staining shows equal loading. (E) A 5-times-more protein input of lysates from ATRA-treated HL60 cells resulted in the detection of a modest increase in Gfi1 protein levels, although the levels are significantly lower compared with monocytic differentiation (see panel D).