Fig 1: RON inhibition downregulates MHC II and CD86 in BMDCs and splenic dendritic cells, which is inconsistent with transcription results for MHC II and CD86. (A−D) On day 9, bone marrow-derived DCs were treated for 6, 12, and 24 h before harvesting, respectively. (A) Representative histogram of flow cytometry analysis of CD86 expression in BMDCs for CD11c+ gated cells. (B) Mean fluorescence intensity (MFI) of CD86 on BMDCs from (A). (C) The expression of MHC II molecules was determined by flow cytometry for CD11c+ gated cells and the value of MHC II shown in the scatter plot represents the percentage of cells. (D) MFI of MHC II on BMDCs from (C). (E, F) DCs were harvested and cultured for 24 h in complete RPMI-1640 medium with stimulation, respectively. The total mRNA was collected with an RNeasy mini column kit and the level of CD86 and CIITA mRNA was tested by RT-PCR. GUSB expression was used as an internal control. Heat map of gene expression for CD86 and CIITA, comparing unstimulated DCs are shown. (G, H) Splenic DCs were isolated from spleen cells of C57/B6 mice and treated with LPS, LPS + MSP + BMS, or LPS + BMS for 24 h, respectively. (G) Representative histogram of flow cytometry analysis of MHC II and CD86 expression in splenic DCs. (H) MFI of CD86 and MHC II from (G). The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS, no significant difference.
Fig 2: (A) IL-1β, IL-12p70, and IL-10 production by LPS-treated BMDCs in the presence of a RON inhibitor leads to downregulation of the ability of BMDCs stimulated with LPS to proliferate T cells. BMDCs were cultured in serum-free RPMI-1640 medium for 2 h and then stimulated with LPS, LPS + MSP, LPS + MSP + BMS777607, LPS + BMS777607, BMS777607 or unstimulated respectively. The supernatant was collected at 24 h and the assay was performed as described in the Methods. The data represent the mean ± SEM of three independent experiments. NS, no significant difference; *p < 0.05; **p < 0.01. (B) The allogeneic MLR protocol was performed as described in the Methods section. The left peak indicates a T cell after proliferation and division. The negative control was CFSE-stained T cells without co-culture with DCs. The experiment was repeated twice independently. (C) Scheme showing that the use of RON inhibitors leads to an increased March-I transcription and thus an increased CD86 and MHC II ubiquitination when LPS stimulated DCs. These changes led to a decrease of IL-1β and IL-12p70 release and increased IL-10 release. Moreover, the ability of LPS-stimulated DCs to stimulate T cell proliferation was decreased when the inhibition of RON.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human MSP/MST1 Protein