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17. PROGRAMAS Y PROYECTOS POR SISTEMAS SISTEMA ECONOMICO
To check if the regulatory role of BAP1 on β-‐catenin is unique to A549 cells or
not, I then explored the consequence of BAP1 depletion in two other human cancer cell lines, namely MCF7 and SW480 cells. MCF7 cell was chosen because I have performed E-‐cadherin related experiments in this cell line (see Chapter 3) and given the known function of E-‐cadherin and β-‐catenin as core components of the AJ, using
the same cell line would allow me to determine the interrelationship between the DUBs idenfied and the two proteins. SW480 cell was chosen because this cell line harbours a truncation mutation in APC, so β-‐catenin is not efficiently degraded and
aberrantly stabilised in this cell line. It would be of therapeutic interest to check if loss of BAP1 function can affect β-‐catenin in this cell line. The siRNA depletion of
BAP1 by all 4 oligos also resulted in decrease in β-‐catenin level in MCF7 cells (Figure
4.4A), indicating that the regulatory role of BAP1 on β-‐catenin was not restricted to
A549 cells only. However, for the case of BAP1 depletion in SW480 cells (Figure 4.4B), only oligo 1 resulted in a significant depletion of β-‐catenin, while the other
oligos did not result in change in β-‐catenin level. Therefore, I did not pursue any
further with SW480 cells.
When observed under microscope, for A549, the control cells (mock transfected or transfected with non-‐targeting siRNA), showed prominent β-‐catenin
staining on the plasma membrane at cell-‐to-‐cell junction and a strong nuclear staining for BAP1 (Figure 4.5A). For cells transfected with the siRNA oligos against BAP1, the nuclear staining of BAP1 was much weaker, indicating the depletion of BAP1. In these cells, the β-‐catenin staining on the plasma membrane was much
weaker, which is in agreement with the biochemical data (Figure 4.3A).
MCF7 cells, which were mock transfected or transfected with non-‐targeting siRNA, were tightly bound to each other with prominent β-‐catenin at cell-‐to-‐cell
junction and strong nuclear staining for BAP1. Similar to A549 cells, the MCF7 cells transfected with siRNA oligos against BAP1 showed less β-‐catenin on the plasma
membrane and weaker nuclear staining of BAP1, indicating BAP1 depletion. Moreover, as seen in Figure 4.5B, cells transfected with oligos 2, 3 and 4 were clearly less tightly bound to each other and more flattened out.
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Figure 4.4. BAP1 depletion in MCF7 and SW480 cells. (A) MCF7 and (B) SW480 cells were transfected using transfection reagent alone (mock) or non-‐targeting siRNA oligo (siControl) or siRNA single oligos against BAP1 (40nM) for 72 hours. Cell were lysed using NP40 lysis buffer 72 hours later. Lysates were resolved on 4-‐12% NuPAGE gel and immunoblotted for β–catenin, BAP1 and actin. Gel images were acquired
by infra-‐red scanner (Odyssey, LICOR).
Figure 4.5. BAP1 depletion decreases β–catenin level on plasma membrane in (A) A549 and (B) MCF7 cells. Cells were transfected using siRNA single oligos against BAP1 at 40nM for 72 hours. After that, cells were fixed in 0.4% PFA/PBS, permeabilised and immunostained with antibody against b–catenin and BAP1. Images were acquired using Nikon microscope and the image setting was the same for all images (Scale bar = 10µm).
A
Figure 4.6. siRNA depletion of BAP1 resulted in decrease in β–catenin mRNA level.
Cells were transfected using siRNA single oligos (40nM) for 72 hours and mRNA was purified using RNeasy Kit. 1µg of mRNA was reverse-‐transcribed and subjected to QPCR analysis. Two biological replicates were analysed and QPCR reactions were set up in triplicate for each biological replicate.
I have also measured the mRNA level of β-‐catenin following BAP1 knockdown
in MCF7 and the experiment was done twice. siRNA depletion of BAP1 using oligos 2, 3 and 4 resulted in a decrease of β-‐catenin mRNA by 20-‐40%, which was consistent
for both biological repeats (Figure 4.6). For knockdown using siRNA oligo 1, at least in one experiment, there was a decrease of β-‐catenin mRNA by about 50%.
When BAP1 was transiently overexpressed in MCF7 cells, there was a reproducible (the experiment was repeated three times), higher level of β-‐catenin
compared to cells transiently overexpressing GFP (Figure 4.7A). This suggested a positive regulatory role of BAP1 on β-‐catenin in MCF7. To assess if this is dependent
on the catalytic activity of BAP1, I repeated the experiment by including two mutant 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -‐ 1 2 3 4 siControl siBAP1
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of BAP1, namely BAP1-‐C91S and BAP1-‐A95D. Both of these mutants lack deubiquitylating activity based on in vitro studies (Ventii et al., 2008). The overexpression of these two mutants did not lead to change in β-‐catenin level, while
the overexpression of wildtype BAP1 resulted in a higher level of β-‐catenin (Figure
4.7B). At face value, it seemed regulatory of BAP1 on β-‐catenin is dependent on its
catalytic activity. However, the expression level of the mutant BAP1 was not as high as that of the wildtype BAP1 and this difference in expression level may account for the lack of effect. In this experiment, I also observed an extra lower molecular weight band below the β-‐catenin band, which was likely a non-‐specific band. This is
because in the two experiments shown in Figure 4.7, the same antibody was used and β-‐catenin always runs slightly below the 97.2kDa marker. Moreover, that was
the band which showed an increase when wildtype BAP1 was overexpressed, which is a very reproducible effect of BAP1 overexpression.
Figure 4.7. Upregulaton of β–catenin following BAP1 overexpression. MCF7 cells
were transfected with 0.5µg of GFP empty vector or 1µg of GFP-‐tagged BAP1, BAP1-‐ C91S and BAP1-‐A95D fusion plasmids. Cell were lysed using NP40 lysis buffer 24 hours later and lysates were resolved on 4-‐12% NuPAGE gels and immunoblottded for β–catenin, GFP and actin. Blot images were acquired by infra-‐red scanner
To further investigate the role of BAP1 on β-‐catenin regulation, I decided to
do a rescue experiment. Before carrying out that experiments, I had to optimise the cell number to be seeded. This is because depletion of BAP1 led to lower number of cells at the end of 72 hours of transfection as evident by the lower protein concentration of lysates obtained (Figure 4.8). The depletion of BAP1 using oligos 1, 2 and 3 led to at least 40% less cells compared to control samples after 72 hours incubation post-‐transfection, whereas the reduction in cell number for oligo 4 was less dramatic. A rescue experiment involves siRNA transfection and DNA transfection, which cause stress to the cells and can lead to extensive cell death. Therefore, more cells should be seeded at the beginning of a rescue experiment to allow enough proteins to be harvested at the end of the experiment for biochemical analysis.
Figure 4.8. Relative protein concentration of MCF7 cell lysates following BAP1 knockdown. MCF7 cells were transfected using non-‐targeting siRNA oligos or siRNA oligos against BAP1 at 40nM for 72 hours. Cell were lysed using NP40 lysis buffer 72 hours later. Bradford protein assay was performed to determine protein concentration of lysates. Data shown represents average of 2 experiments.
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-‐ OL1 OL2 OL3 OL4 siC siBAP1