ejecutada simultaneamente con las obras de urbanización y construcción.
NORMAS SOBRE VOLUMETRIA PARAMENTOS Y LINDEROS
3.2.4 Identification of DUBs regulating E-‐cadherin in A549
Two colleagues in our laboratory, Joseph Sacco and Han Liu, performed a large scale DUB library screen in A549 cells and the NP40 cell lysates were processed for SDS-‐PAGE and aliquoted into 96-‐well plates, serving as a resource for the identification of DUBs playing roles in regulating a cellular process or stability of a protein in steady state conditions (Figure 3.15). I have utilised this resource as a parallel mean to identify DUBs involved in E-‐cadherin regulation.
Figure 3.15. Large scale DUB siRNA library screen. A549 cells were seeded in 15cm dish and cells were either mock-‐transfected or transfected with non-‐targeting siRNAs or pool of 4 single oligos against 1 of 92 DUBs, at a final concentration of 40nM. Cells were lysed using NP40 lysis buffer 72 hours post-‐transfection and following protein assay, lysates were boiled with sample buffer and adjusted to a concentration 0.8µg/µl. Finally, 25µl of each knockdown samples was arrayed on 96-‐ well plates. (experiment was performed by Liu and Sacco)
Knockdown of 96 DUBs using a DUB siRNA library from Qiagen
72hrs KD Protein
Harvest
Short spin
Sample buffer added to give samples of 1mg/ml
Boil 98oC for
10 min
Pipette each sample into appropriate well
Transfer 25ul of each sample and control into a 96 well plate using multichannel pipette
Figure 3.16. siRNA DUB library screen to identify DUBs regulating E-‐cadherin in A549. NP40 lysates from large scale DUB screen performed by Sacco and Liu were resolved on 10% SDS-‐PAGE gels, followed by Western Blotting. Blots were probed with antibodies against E-‐cadherin, tubulin and actin. Gel images were acquired by an infra-‐red scanner (Odyssey, LICOR). Notes : Control = mock knockdown without oligo; siControl1 = knockdown with non-‐targeting oligos.
Figure 3.16 shows the representative blot image acquired for this set of experiments. At first glance, depletion of a number of DUBs on Gel1 resulted in visible increase in E-‐cadherin level as compared to the controls. Across all the gels, TNFAIP3, USP27X and USP49 resulted in the most dramatic increase in E-‐cadherin level.
Densitometric analysis of the blot images was performed to give a quantitative measurement of the E-‐cadherin level (Figure 3.17). The top DUB candidates, which when silenced resulted in significant increase in E-‐cadherin level,
as determined by both quantitation approaches, were TNFAIP3, USP27X, USP49 and USP42, in agreement with the visual observation. On the other end of the graph were ZRANB1, OTUB2, USP38 and PAN2, which when silenced resulted in depletion of E-‐cadherin. OTUD6A did not appear in the top hit lists, although it was quite close to the negative end.
Figure 3.17. Change in E-‐cadherin level following knockdown of individual DUBs. Densitometric analysis was performed using ImageJ to determine relative amount of E-‐cadherin for each knockdown sample and E-‐cadherin level was normalised to (A) tubulin or
(B) actin. The percentage change in E-‐cadherin level relative to that of the median sample was calculated and log-‐transformed. Data shown represents average of 3 technical repeats and error bars represent standard deviation.
A
Figure 3.18. Summary of DUBs whose knockdown result in change in E-‐cadherin level. Summary was based on results of siRNA DUB library screen performed in MCF7 and A549 (presented in Figure 3.13 and Figure 3.17 respectively). Only DUBs, which are identified as top hits by both quantification results are listed. DUBs whose knockdown resulted in increase in E-‐cadherin level are highlighted in red while those resulted in decrease in E-‐cadherin level are highlighted in black.
Figure 3.18 summarises the top hits, based on their effect on E-‐cadherin stability, from the two separate siRNA DUB library screens in MCF7 and A549 respectively. USP38 is the only DUB which when siRNA depleted results in decrease E-‐cadherin level, while there was no overlapping candidates for DUBs which when siRNA depleted results in increase in E-‐cadherin level.
Figure 3.19. Deconvolution of targets which resulted in change in E-‐cadherin level in A549. (A) A549 cells were transfected with single oligos or pool oligos against candidate DUBs at a final concentration of 40nM. Cells were lysed using NP40 lysis buffer, and lysates were resolved by SDS-‐PAGE and immunoblotted for E-‐cadherin. Gel images were acquired by an infra-‐red scanner (Odyssey, LICOR). Arrows indicate direction of change of E-‐cadherin following knockdown of corresponding DUBs in the screen. (B) Densitometric analysis was performed using Image J software to determine E-‐cadherin band intensity and normalised to actin level. Change in total E-‐ cadherin level from that of non-‐targeting controls was then determined and represented in the graph.
-‐80 -‐60 -‐40 -‐20 0 20 40 1 2 3 4 P 1 2 3 4 P 1 2 3 4 P 1 2 3 4 P 1 2 3 4
USP27X USP49 TNFAIP3 siUSP38 OTUD6A
A B Percen ta ge ch an ge i n To ta l E -‐ca dh eri n l evel
Among the targets identified in this screen, TNFAIP3, USP27X, USP49, USP38 and OTUD6A were chosen for deconvolution (Figure 3.19). While the first three candidates were chosen for the dramatic increase in E-‐cadherin following their depletion, the latter two were chosen because USP38 depletion resulted in the decrease in total E-‐cadherin level while OTUD6A depletion decreased the full length E-‐cadherin to 80kDa fragment ratio in MCF7 cells. For both USP27X and USP49, the repeat knockdown using pool oligos did not reproduce the dramatic increase in E-‐ cadherin level that was observed during the screen. All the individual oligos against USP27X and 3 out of 4 oligos against USP49 did not result in any increase, but decrease, in E-‐cadherin level either. For TNFAIP3, oligo 2 and 4 recapitulated the pool knockdown effect, resulting in increase in E-‐cadherin level, but not to the extent that was observed with the screen. While 2 oligos giving the same effect as the pool oligos is sufficient to eliminate the possibility of off-‐target effects, the immunoblot to assess knockdown efficiency of TNFAIP3 suggested otherwise. Firstly, oligo 4 did not effectively silence TNFAIP3 and interestingly, the other 2 oligos, which had higher silencing potency, in fact resulted in decrease in E-‐cadherin level. For USP38, the decrease in E-‐cadherin level following transfection of the pool oligos was quite mild, and only oligo 3 resulted in a more dramatic loss of E-‐cadherin. 2 out of the 4 oligos against OTUD6A resulted in a slight decrease in E-‐cadherin level. Therefore, the deconvolution results for USP38 and OTUD6A in USP38 were not convincing enough to indicate a functional role between the DUBs and E-‐cadherin in A549 cells.