In order to assess the biological activities of ephrinB2, ephrinB2/G or ephrinB2/ΔV, we needed the expression levels of the mutants to be comparable with that of the wild type protein. Therefore, we decided to generate tetracycline inducible MCF7 cell lines which expressed ephrinB2 wild type or the mutants only in the presence of the antibiotic tetracycline (or doxycycline). This system presented us with the opportunity of growing the newly generated MCF7 cell lines under standard conditions and actively regulating the expression of the proteins at our discretion. We decided to use the MCF7 TetOn cells (purchased from Clontech). According to the supplier’s manual, these cells are stably transfected with the pTet-On Advanced vector which encodes the Tet-On Advanced transcriptional activator. This engineered transcription factor consists of a mutant E. coli TetR protein (rTetR) fused to three minimal “F”-type activation domains derived from the herpes simplex virus (HSV) VP16 protein. In the presence of doxycycline, Tet-On Advanced binds to the tetO sequences in the
PTight inducible promoter on a different expression vector (in our case, the pBI vector)
containing the gene of interest and induces gene expression. As PTight lacks binding sites for
endogenous mammalian transcription factors, it is virtually silent in the absence of induction by doxycycline. Before generating our cell lines of interest, we established that the levels of endogenous EphB4 and syntenin-1 were comparable to those seen in our MCF7 cells. We also found that like MCF7 cells, MCF7 TetOn cells did not express ephrinB ligands (data not shown). We then stably transfected MCF7 TetOn cells with the tetracycline inducible pBI plasmid encoding either the wild type or the ephrinB2 mutants. Tetracycline-dependent expression was initially tested by flow cytometry (data not shown). Cells were subsequently
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sorted by FACS using anti-Flag mAb for high expression of wild type or ephrinB2 mutants on the cell surface 48 hours after stimulation with doxycycline. We conducted FACS sorting twice in order to obtain a homogenous cell population expressing high levels of ephrinB2 upon doxycycline stimulation. Newly generated cell lines were called MCF7 Tet-ephrinB2, MCF7 Tet-ephrinB2/G and MCF7 Tet-ephrinB2/∆V. We also generated a MCF7 Tet- ephrinB2 cell line in which we depleted endogenous syntenin-1 using a lentiviral-based approach. After testing five different commercial shRNA lentiviral constructs targeting syntenin-1 in MCF7 TetOn cells (data not shown), we selected the most effective and used it to generate the MCF7 Tet-ephrinB2/shSyntenin-1 cell line. As shown in figure 3.7C, we managed to achieve ~75% knock down of endogenous syntenin-1 in these cells.
In order to characterize the newly established tetracycline inducible cell lines, we performed flow cytometry analysis and immunofluorescence staining using anti-Flag mAb. We also verified expression levels of ephrinB2 and syntenin-1 by Western blotting. For all experiments, we analyzed cells before and after stimulation with doxycycline. In figure 3.7A, the histogram shows cell surface expression levels of ephrinB2 and mutants. These results are presented as percentages of ephrinB2 mean fluorescence intensity upon doxycycline stimulation (considered as 100%). These data are also presented in table format in figure 3.7B with negative and positive control values. Unstimulated cells expressed low levels of ephrinB protein on the cell surface (blue columns), which was expected, as inducible expression systems are subject to gene expression leakiness. Expression levels of ephrinB2/G and ephrinB2/ΔV after doxycycline stimulation (red columns), were ~20% lower than ephrinB2. However, in syntenin-1-depleted MCF7 Tet-ephrinB2 cells, ephrinB2 expression was ~30% higher. Interestingly, we noticed that cell surface expression of tetraspanin CD81 (ie the positive control) was also notably higher in syntenin-1-depleted cells.
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The overall protein expression levels of ephrinB2/G and ephrinB2/∆V (lanes 4 and 6 respectively) where slightly lower than ephrinB2 (lane 2) when measured by Western blotting (figure 3.7C). As expected, syntenin-1 levels were considerably lower in syntenin-1-depleted cells (lanes 7 and 8). β-actin levels were also measured and served as internal loading control. In order to obtain accurate values of overall expression levels, which we would be able to compare to flow cytometry values, we performed densitometric analysis of Western blots and presented the results as percentages of the expression level of ephrinB2 wild type upon stimulation with doxycycline (taken as 100%) (figure 3.7D). Our results show that the difference in cell surface expression observed for ephrinB2/G and ephrinB2/ΔV were probably caused by differences in overall expression, as the two mutants are expressed at ~20% lower levels than ephrinB2. Interestingly, the total ephrinB2 expression level in the presence of syntenin-1 knock down was ~10% lower than that in non-depleted cells.
Immunofluorescence staining of ephrinB2, ephrinB2/G and ephrinB2/∆V proteins using anti-Flag antibody confirmed that PDZ binding motif mutations and syntenin-1 depletion did not alter cell surface localization (figure 3.7E). As in the stable MCF7-ephrinB2 cell line, stronger staining was observed on the plasma membrane. However, in MCF7 Tet- ephrinB2/shSyntenin-1 cells ephrinB2 staining appeared to be more diffuse.
In summary, these experiments confirmed that the tetracycline-inducible system worked and that stimulation with 1 µg/ml doxycycline for 48 hours was sufficient to ensure high levels of protein expression in all four cell lines. More importantly, all cell lines expressed comparable levels of ephrinB2 proteins, albeit with minor differences.