Fig. 5: Effect of FCN2 inhibitors on complement activation by NPs in PS and on NP uptake by phagocytic cells.

a TMT shotgun analysis of the effect of GlcNAc (100 mM) and DMNAc (100 mM) on the proteome of PMOXA-coated NPs incubated in PS. The graphs represent identified proteins in order of % increasing abundancy in agonist-treated NPs relative to control NPs. FCN2 (green) and major complement proteins (blue) are indicated. Source data are provided as Source Data file. b Analysis of the amount of indicated proteins bound to PMOXA- and PEOXA-coated NPs, identified by TMT shotgun proteomics after incubation in PS in the presence of the indicated agonists, expressed as % of controls. GlcNAc, Man, and Gal were used at 100 mM concentration; EDTA was 10 mM. DMNAc and DMNPr were used at the concentration of 100 mM with PMOXA-coated NPs and at the concentration of 300 mM with PEOXA-coated NPs. The data shown is the mean ± range (n = 2). Source data are provided as Source Data file. c TMT-based relative quantification of total C3, C3b, and iC3b bound to PMOXA-coated NPs, using fragment-specific peptide markers (see scheme), after incubation in PS in the presence of GlcNAc and DMNAc (100 mM). Data were the mean ± range (n = 3). Differences with controls were not significant (p > 0.05) as determined by two-way ANOVA with Šidák’s multiple comparison tests. Source data are provided as Source Data file. d, e Effect of sugar and amide FCN2 inhibitors on NP uptake by pig phagocytes NPs. NPs were pretreated with PS alone or in the presence of indicated agonists (GlcNAc and Man 100 mM for both PMOXA-NPs and PEOXA-NPs; DMNPr and DMNAc 100 mM for PMOXA-NPs and 300 mM for PEOXA-NPs), for 20 min at 37 °C. NPs were washed and further incubated in sera and agonist-free RPMI with pig macrophages (d) and pig monocytes (e). After 3 h cells were washed and analysed by FACS. NPs uptake was based on normalized MFI and expressed as % of control cells (Ctr; no agonists). GlcNAc N-acetyl-d-glucosamine, Man mannose, DMNPr N,N-dimethylpropionamide, DMNAc N,N-dimethylacetamide. Values are means ± SD (macrophages, n = 5 independent experiments; monocytes n = 4–8 independent experiments. Significant p values (<0.05), calculated by two-way ANOVA with Šidák’s multiple comparison test. f, g Data represent uptake of PMOXA-coated NPs by human macrophages and monocytes, respectively (G) in the presence of HS with and without the indicated agonists (100 mM). Values are MFI means ± SD (macrophages, n = 12 independent experiments, monocytes n = 6 independent experiments). Significant p values (<0.05), calculated by two-way ANOVA with Šidák’s multiple comparison tests. Source data are provided as Source Data file.