Article Title: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome
Figure Lengend Snippet: ( A – D ) Examining the effect of proteasome subunit knockdown in different cell lines. 80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 ( A ), H838 ( B ), T47D ( C ), and H1792 ( D ) cells by viral transduction. Each subunit was targeted by 4 different shRNAs and the cell viability was measured as the relative cell number compared to the average of non-targeting shRNAs 5 days after the initial introduction of the shRNAs. ( E , F ) HepG2 cells with shRNAs targeting PSMC5, PSMD2, and green fluorescent protein (GFP) were grown out. Their relative growth was analyzed in the absence of bortezomib ( E ) and their protein content was analyzed 24 hr after the addition of either 8 or 12 nM of bortezomib ( F ). ( G ) The relative cell number of cells harboring a control shLacZ (black) or each of 5 individual shRNAs targeting shPSMC5 (Cayenne) was analyzed 4 days after addition of the indicated concentrations of bortezomib. ( H ) HepG2 cells stably expressing shRNAs targeting the PSMC5 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. ( I – N ) The HepG2 cells with shRNAs targeting PSMC5, PSMD2, and GFP (described above) were further exposed to a short panel of stress inducers including bortezomib ( I ), tunicamycin ( J ), rohinitib-RHT ( K ), Hsp90 inhibition ( L ), withaferin A ( M ), and cyclohexamide ( N ) at indicated concentrations and the relative cell number (RFU) was examined after 4 days. The graphs represent the average of at least 4 different measurements and the SEM. DOI: http://dx.doi.org/10.7554/eLife.08467.006
Article Snippet: MG132 (EMD Millipore, Billerica, MA, United States), Bortezomib (LC Laboratories # B-1408, Woburn, MA, United States), Cyclohexamide (Enzo life sciences, Farmingdale, NY, United States), withaferin A, tunicamycin (Sigma St. Louis, MO, United States).
Techniques: Transduction, Stable Transfection, Expressing, shRNA, Western Blot, Inhibition