Fig 2: Phosphorylation of SCIMP at Y96 enhances binding to TLR4.(a) Pull-down assays using LPS-activated RAW264.7 cell lysates and GST-tagged tyrosine to phenylalanine (Y to F) mutants of SCIMP-T1. SCIMP interactions with either TLR4 or Lyn were assessed by immunoblotting. (b) GST pull-down assays were performed using GST-SCIMP mutants and recombinant His-tagged TLR4-TIR domain, and binding was evaluated using immunoblotting against His-TLR4-TIR (upper panel). Quantification of immunoblots was performed using densitometry. Data are combined from two independent experiments (mean+range, lower panel). (c) Phosphorylation at Y96 enables SCIMP to bind to TLR4. Titration of fluorescently labelled p-Y96-containing (red) or Y96-containing (green) SCIMP-derived peptides (aa 90–107) with TLR4-TIR. Inset shows fluorescence recovery over a longer time course with unlabelled p-Y96 peptide. (d) The Kd (159 nM and>2.5 µM for p-Y96- and Y96-containing peptides, respectively) was determined by curve fit analysis. (e) SCIMP phosphorylation at Y96 is required for in vivo TLR4 binding. RAW264.7 cells stably expressing wild type (WT) or Y96F SCIMP were used to assess the agonist-induced SCIMP-TLR4 interaction, by co-immunoprecipitation of SCIMP followed by immunoblotting for TLR4 and SCIMP (left). The level of TLR4 pull-down was quantified relative to SCIMP levels (right) to correct for minor differences in levels of WT versus Y96F SCIMP expression (mean+s.e.m.). Data in a are representative of three independent experiments, data in b are representative of (top) or combined from (bottom) n=2 independent experiments, and data in e are representative of three independent experiments (left) or combined from n=3 independent experiments (right).

Fig 3: Effect of corilagin on the TLR4 signaling pathway after ox-LDL stimulation in MPMs. (A) mRNA expression of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO, and IRF5 was measured by qRT-PCR. *P < 0.05 compared with the control group, #P < 0.05 compared with the ox-LDL group, **P < 0.05 compared with the aspirin group determined by one-way ANOVA and subsequent S–N–K method (n = 5). (B,C) Protein expression was measured by Western blotting. *P < 0.05 compared with the control group, #P < 0.05 compared with the ox-LDL group, **P < 0.05 compared with the aspirin group determined by one-way ANOVA and subsequent S–N–K method (n = 5).

Fig 4: MSCs from different genetic backgrounds have distinct antibacterial properties(A) Cartoon depicting the antibacterial assays used in this study. MSCs were incubated overnight with or without lipopolysaccharide (LPS) from E. coli (100 ng/mL) and were then co-cultured with E. coli strain K12 for 6 h. After 6 h of co-culture, E. coli abundance in the media was quantified using colony-forming unit (CFU) assays.(B) Quantification of E. coli CFUs following 6-h co-culture with different cell types +/- LPS treatment. E. coli CFU values from co-culture experiments were normalized to controls in which E. coli was grown side-by-side in monoculture (n = 5 biological replicates per condition; statistical significant determined using t test; *p < 0.05; **p < 0.01; individual data points are shown and bar graphs represent the mean with error bars = SEM).(C and D) Cytokine profiling of C57-MSC and BALB-MSC conditioned media with or without LPS-priming using BioLegend LEGENDplex bead-based immunoassays with the mouse inflammation panel or (D) proinflammatory chemokine panel. Samples were analyzed 18-h post-LPS-exposure, and the values shown on heatmap are the mean of at least two biological replicates.

Fig 5: Effect of corilagin on expression of molecules involved in the TLR4 signaling pathway after ox-LDL stimulation in TLR4-upregulated Ana-1 cells. (A,B) Upregulation of TLR4 expression in Ana-1 cells via the lentiviral vector. GFP expression was observed with a fluorescence microscope after the lentivirus had been introduced into Ana-1 cells for 72 h. (C) After elimination by puromycin for 7 days, fluorescence was observed. (D) Expression of TLR4 mRNA was measured by qRT-PCR. *P < 0.05 compared with the control group determined by the Student's t-test (n = 5). (E,F) Expression of TLR4 protein was measured by Western blotting. *P < 0.05 compared with the control group determined by the Student's t-test (n = 5). (G) mRNA expression of molecules involved in the TLR4 signaling pathway was measured by qRT-PCR. *P < 0.05 compared with the lentivirus-TLR4 group, #P < 0.05 compared with the model group (lentivirus-TLR4 + ox-LDL group), **P < 0.05 compared with the aspirin group determined by one-way ANOVA and subsequent S–N–K method (n = 5). (H,I) Abundance of the protein of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO, and IRF5 was measured by Western blotting. *P < 0.05 compared with the lentivirus-TLR4 group, #P < 0.05 compared with the model group, **P < 0.05 determined by one-way ANOVA and subsequent S–N–K method (n = 5).