To assess the sensitivity of the PIR cell-based assay, H1299-PIR cells were incubated for 8 hours with different concentrations (0.01 10 µM) of known proteasome inhibitors (MG132 and Bortezomib).
Moreover, the potency of proteasomal inhibition, judged by these criteria, coincides nicely with the magnitude of the nuclear fluorescence signal detected in the PIR cell-based assay, upon inhibition with the different hit compounds.
The ligature was introduced into wild-type P. temperata cells by conjugation with donor E. coli S17-1 lambda pir cells.
PIR cells were plated onto 384-well microplates for 24 hours, and then treated for 48 h with the library compounds.
Further characterization of the new inhibitors discovered using this approach, as well as the development of a combined ultra high resolution / high content screen, based on the PIR cells, are currently underway.
Towards this end, MDM2, a p53 E3 ubiquitin ligase and a known target of proteasome-dependent degradation, was transfected into PIR cells, and its localization was assessed by immunofuorescence microscopy.
pDS132-based allelic exchange plasmids [ 23] were electroporated into E. coli S17-1λ pir cells for conjugation into E. coli K92 by filter mating [ 29].
To check whether MDM2 expression is critical for PIR nuclear translocation, we performed siRNA-mediated knockdown of MDM2 expression in PIR-cells, and then treated the cells with proteasome inhibitors (Figure 2b).
Thus, H1299-PIR cells appear to be sensitive reporters, capable of detecting the activity of proteasome inhibitors in a cell-based assay.
For this purpose, E. coli S17-1 λ-pir (pMiniHimaRB1B1) (E. coli S17-1 λ-pir carryingrrying plasmid pMiniHimar RB1) was used as a donor in mating with Ech3937 [39].
H1299-PIR cells were lysed with radioimmune precipitation assay buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0) containing a protease inhibitor cocktail from Roche Applied Science.
