Fig. 2: Priming with IL-1β^low impairs cytokine-triggered NF-κB pathway activation in INS-1E cells.

A–C INS-1E cells were preconditioned with IL-1β^low and then challenged or not with CYT. After indicated time, protein levels of phospho-IκBα and total-IκBα were analyzed by Western blot. β-actin was used as a loading control. Data are shown as mean ± SEM, n = 5. D, E INS-1E cells were treated as described in (A), followed by 30 min stimulation with CYT. Cellular localization of p65 NF-κB immunostaining (red) was analyzed by fluorescence microscopy; nuclei were counterstained with Hoechst (blue). Scale bars: 10 μm. Nuclear-to-cytoplasmic ratio was quantified based on analysis of ten high-power fields per condition, n = 4. F IL-1β^low-preconditioned INS-1E were transiently transfected with κB-LUC and CMV-RL reporter plasmids. After 24 h, cells were challenged or not with CYT for 16 h. Firefly luciferase (LUC) activity was then measured and normalized to Renilla luciferase (RL) activity as control for transfection efficiency, n = 3. G IL-1β mRNA and H) IL-1Ra mRNA levels were assessed by RT-qPCR in IL-1β^low-preconditioned INS-1E cells, with or without CYT challenge for 6 h. Relative mRNA levels were normalized to HPRT, n = 3. Data are shown as mean ± SD. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.