INS-1 and INS-1E cells (generously provided by Claes Wollheim and Pierre Maechler, University of Geneva, Switzerland) were maintained as previously described (Storling et al. 2005 (link); Chou et al. 2012 (link)). Cells were mycoplasma negative. Human pancreatic islets were isolated by collagenase digestion, density gradient purification and hand-picking from the glands of 16 organ donors (for donor characteristics, see Table 1 ) and then cultured in M199 culture medium, supplemented with 10% bovine serum and antibiotics in a CO2 incubator at 37°C as described previously (Bugliani et al. 2013 (link)). No gender related differences were noted for any outcome variable, and data are therefore combined.
Recombinant rat (rr) IL-1β and recombinant mouse (rm) TNFα were purchased from R&D Systems (Minneapolis, USA). Rm IFNγ and Griess reagent were purchased from Sigma (St Louis, MO, USA). Recombinant human (rh) IL-1β and IFNγ were purchased from Roche Diagnostics (Mannheim, Germany). Trypsin-, chymotrypsin- and caspase-like proteasomal activity reagents were purchased from Promega (Madison, Wisconsin, USA). The broad proteasome inhibitor MG132 and the selective immunoproteasome inhibitor ONX-914 (200 nM), 20–40- fold more selective for Psmb8 than Psmb9 (Muchamuel et al. 2009 (link)), were from Selleckchem (Houston, Texas, USA). The histone deacetylase (HDAC)1–3 inhibitor MS-275 was from Selleckchem, whereas the inhibitor of the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) BRD0476 that in turn highly selectively inhibits Janus activated kinase(JAK)/signal transducer and activator of transcription (STAT)1 and the HDAC1-2 selective ‘3 ‘inhibitor were synthesized in-house (Chou et al. 2012 (link); Chou et al. 2015 (link)).
Recombinant rat (rr) IL-1β and recombinant mouse (rm) TNFα were purchased from R&D Systems (Minneapolis, USA). Rm IFNγ and Griess reagent were purchased from Sigma (St Louis, MO, USA). Recombinant human (rh) IL-1β and IFNγ were purchased from Roche Diagnostics (Mannheim, Germany). Trypsin-, chymotrypsin- and caspase-like proteasomal activity reagents were purchased from Promega (Madison, Wisconsin, USA). The broad proteasome inhibitor MG132 and the selective immunoproteasome inhibitor ONX-914 (200 nM), 20–40- fold more selective for Psmb8 than Psmb9 (Muchamuel et al. 2009 (link)), were from Selleckchem (Houston, Texas, USA). The histone deacetylase (HDAC)1–3 inhibitor MS-275 was from Selleckchem, whereas the inhibitor of the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) BRD0476 that in turn highly selectively inhibits Janus activated kinase(JAK)/signal transducer and activator of transcription (STAT)1 and the HDAC1-2 selective ‘3 ‘inhibitor were synthesized in-house (Chou et al. 2012 (link); Chou et al. 2015 (link)).
