Proteasomal Gfi1 degradation is diminished during differentiation. (A) U937 cells were differentiated with PMA and collected at the indicated time points. Cell lysates were normalized using Bradford protein quantification assay and used in an in vitro degradation assay with 35 S-labeled in vitro–translated full-length Gfi1. (B) Differentiated and undifferentiated U937 cells were treated overnight with MG132. Endogenous Gfi1, ubiquitin, and lamin levels were quantified by Western blot. Note the clear increase in Gfi1 levels in undifferentiated U937 cells after proteasome inhibition. Lamin staining was used as a control for equal loading. Ubiquitin staining was performed to confirm the global effectiveness of MG132. (C) In undifferentiated HL60 cells proteasome inhibition results in an accumulation of Gfi1 protein levels. The same controls were used as in panel B. (D) The stability of endogenous Gfi1 was studied in undifferentiated and 48-hour PMA-treated U937 cells. Cells were treated with 25 μg/mL cycloheximide for the indicated times. Lamin staining was used to check for equal loading and the density of Gfi1 bands was quantified using ImageJ software. The relative protein amount was plotted against the time of cycloheximide treatment. ▴ indicate the relative amount of Gfi1 in untreated cells and ▪ indicate the relative amount of Gfi1 in 48-hour PMA-differentiated cells. (E) 26S proteasome function in lysates from differentiated U937 cells was analyzed by measuring the cleavage of Suc-Leu-Leu-Val-Tyr-AMC in the lysate for 2 hours at 37°C. (F) U937 cell lysates were immunoblotted and stained for ubiquitin. Cell lysates treated overnight with MG132 were used as a positive control for decreased proteasome activity. Equal loading was confirmed with actin staining. (G) In vitro–translated wild-type Gfi1 (WT), and the Gfi1 point mutants A382S and K403R were used in an in vitro degradation assay with 72-hour ATRA-treated and untreated U937 cell lysates for the indicated time points. Cell lysates were normalized using the Bio-Rad protein quantification assay.
