Fig. 3.

Iminosugars dampen the response signalled by IFNγ receptor and TNFα receptor. Primary human MDMΦs matured in the presence of IL-4 were treated with iminosugars for 2 days (100 μM NB-DNJ or 25 μM MON-DNJ) then stimulated with either (A) 250 pg/ml recombinant human IFNγ (R&D Systems) or (B) 250 pg/ml TNFα (Peprotech), or mock stimulated (no treatment). RNA was extracted from the cells 6 h later (Direct-zol™ RNA Mini-prep kit (Zymo Research)) and RT-qPCR performed (using the Verso 1-step RT-qPCR kit in a volume of 20 μl according to the manufacturer's instructions on an Applied Biosystems 7500 real-time PCR system) for (A) CXCL10 (n = 4 donors cells) or (B) TNFAIP3 (n = 3 donors cells). TaqMan Gene Expression Assays containing gene-specific probe and primer sets were used for quantitative gene expression analysis of CXCL10 (assay ID Hs01124252_g1) and TNFAIP3 (assay ID Hs00234713_m1), ribosomal protein lateral stalk subunit P2 (RPLP2) (assay ID Hs01115128_gH) was also amplified and used as an endogenous control (all Thermo Fisher). 2 μl of each sample was analysed in technical duplicate and RNA quantified by the ΔΔCt method. The relative quantity of RNA is shown for individual donors, normalised to an RPLP2 endogenous control, mean and SD. A two-way ANOVA comparing between treatments using Holm-Šidák correction for multiple comparisons was performed and those treatments with significant changes in comparison with levels produced by cytokine stimulus in the absence of iminosugar are highlighted with asterisks. *p < 0.05, **p < 0.005, ***p < 0.0005.