Figure 5. Interaction between BTG2 APRO and PABPC1 RRMs is necessary for BTG2 APRO domain to reduce cell proliferation.

(a) Histograms showing the cell proliferation dye intensities in cells expressing or not BTG2(APRO). U2OS cells were labelled with the cell proliferation dye eFluor 670 before seeding in transfection plates. One day later, the cells were co-transfected with plasmids expressing GFP and BTG2(APRO)-HA (right panel) or GFP and empty expression vector (left panel), and were analysed 3 days after transfection using flow cytometry. (b) Histogram representing generation time of cells expressing or not the APRO domain of BTG2. U2OS cells were treated as in a and analysed 48, 72 and 96 h after transfection using flow cytometry. Half-life for the dilution of the mean proliferation dye intensity for the different cell populations was calculated from kinetic curves. These half-lives correlate directly to the cell-generation doubling time. Error bars (s.d.) correspond to two biological replicates. (c) Analysis of the ability of mutated BTG2 APRO domains to reduce cell proliferation. The analysis depicted in a was repeated with plasmids expressing WT, boxC-mutated (same mutation as in Fig. 3a) or 71+ insertion-mutant BTG2 APRO domains, or empty expression plasmid as control. Proliferation rate changes, quantified from the eFluor670 dye dilution, are plotted according to the formula: Proliferation index=1− [(mean dye intensity for GFP-positive cells–average of mean dye intensities for GFP-negative cells from all samples)/average of the mean dye intensities for GFP-negative cells from all samples]. Hence, a value of 1 corresponds to the proliferation of fast-growing GFP-negative cells, while an index of 0 indicates a reduction of one cell cycle division after transfection. Average (±s.d.) from two biological replicates. (d) A western blot analysis of cell lysates corresponding to the experiment presented in c is shown to monitor expression of the BTG2 proteins.