Fig. 3

Gfi1 overexpression enhances cell proliferation and survival and protects MM cells from Btz-induced apoptosis. MM.1S cells were transduced with lentivirus carrying the PCL6-GFP vector containing the Gfi1 cDNA insert (Gfi1 o/e) or the empty vector (EV). Gfi1 overexpression was estimated at both mRNA as measured by qPCR (a, left panel) and protein levels as measured by WB (a, middle panel) and quantified (N = 9) by densitometry (a, left panel). Stable Gfi1 o/e MM.1S cells and their EV controls were stained with CellTracker, and proliferation was measured after 72 h by flow cytometry (b, histograms). The bar graph represents the fold increase in proliferation of Gfi1 o/e cells as compared with EV in three independent experiments (b). Cultures of MM.1S cells, Gfi1 o/e, and EV controls (24 h) were stained with propidium iodide (PI), and cell cycle phases were evaluated by flow cytometry (c, histograms). The bar graph represents the percent of cells in the “G2+M” cell cycle phase of Gfi1 o/e cells compared with EV in three independent experiments (c). MM.1S Gfi1 o/e and EV control cells were treated with Btz at the indicated concentrations. Viability was measured by alamarBlue assay after 24 and 48 h and analyzed as percent from the untreated control (d). MM.1S EV and Gfi1 o/e cells were treated for 24 h with Btz (3 and 5 nM), and cell lysates were analyzed by WB for Gfi1 and total and cleaved caspase 3 protein levels using β actin as loading control (e)