Concanamycin a

MG132, Z-VAD-FMK (Selleckchem); MLN4924, GW4869 (Sigma); chloroquine (Invivogen); concanamycin A, rapamycin (Enzo); spiroepoxide (Santa Cruz); staurosporine (CST). Recombinant glycosidases PNGaseF and EndoH, and the substrate recombinant RNaseB protein were purchased from New England Biolabs.

Human embryonic kidney HEK293 cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. HEK293-NSP1 stable cell lines were generated from HEK293 cells and cultured in complete DMEM in the presence of puromycin (0.5 μg/ml). Expression of NSP1 was induced by doxycycline (1 μg/ml) treatment for 24 hr. African Green Monkey kidney MA104 cells were obtained from ATCC and cultured in Medium 199 supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Cells were stimulated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), IFN-β (100 U/ml), poly (I:C) LMW/LyoVec (100 ng/ml) for 15 min or 6 hr for either western blot/immunofluorescence or QPCR quantification, respectively. Cells were treated with either small-molecule proteasome inhibitors (10 μM) for 12 hr: MG-132, bortezomib, carfilzomib, VR23, celastrol, curcumin from Selleckchem, lactacystin and epoxomicin from Enzo Life Sciences; or lysosome inhibitors (10 μM) for 12 hr: chloroquine (InvivoGen), concanamycin A (Enzo), bafilomycin A (Sigma). NEDD8 activating enzyme inhibitor MLN4924 (Millipore) was reconstituted in DMSO (stock concentration: 20 mM) and used at the range of 100 nM to 10 μM.

To obtain co-knock of V-ATPase and SOX11, Z138 SOX11^IND+/SOX11^IND- cells were transfected using an Amaxa 4D-Nucleofector (Lonza, Basel, Switzerland). Cells, 2.5 million were resuspended in SF solution (Lonza) and mixed with 500 nM pool of siRNA (Sigma-Aldrich) targeting V-ATPase. In each reaction a scrambled sequence (SCR) was used as a control.
After transfection, 50,000 cells were plated out in 96 well Cytostar-T plates, and grown for 24 h, before treated with different concentrations of bafilomycin A1 and concanamycin A (Enzo Life Sciences), respectively, for up to 48 h. Cell proliferation, by incorporation of [14C]-thymidine, was measured as described above.