Fig. 5: IL-1β^low boosts cytokine-induced eIF2α phosphorylation promoting cellular stress adaptation.

INS-1E cells were preconditioned with IL-1β^low and subsequently challenged or not with CYT for 16 h. A, B Protein levels of phospho-eIF2α and total eIF2α were analyzed by Western blot and quantification expressed as phospho-eIF2α/total eIF2α ratio. β-actin was used as a loading control, n = 4. C–G Protein levels of BIP, GRP94, ORP150 and PDI were analyzed by Western blot; β-actin was used as loading control, n = 3-5. H BIP mRNA expression was analyzed by RT-qPCR and relative mRNA levels normalized to HPRT; a 24 h washout condition (without CYT) was also evaluated, n = 4. I XBP1 mRNA splicing was analyzed by RT-qPCR and expressed as XBP1s/XBP1t mRNA ratio. Relative mRNA levels were normalized to HPRT, n = 5. J, K IL-1β^low-preconditioned INS-1E cells were transiently transfected with either the 5×ATF6-LUC or XBP1u-LUC reporter plasmids, along with the CMV-RL plasmid. At 24 h post-transfection, cells were challenged or not with CYT for 16 h. Firefly luciferase (LUC) activity was measured and normalized to Renilla luciferase (RL) activity, n = 4. Tunicamycin (2 µg/mL, Tn) and thapsigargin (50 nM, Tg) for 16 h were used as positive controls. Data are presented as mean ± SD. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.