Fig 1: Liver-specific RACK1 deficiency leads to MDSC accumulation under steady-state conditions. (A) 12-week-old Rack1?hep mice and littermate controls were injected with LPS/GalN. After the indicated time periods, livers were isolated and subjected to microarray analysis. Genes involved in the recruitment and local expansion of MDSCs are shown in heatmaps. Mean values from two mice are shown. (B, C, E) Livers of 12-week-old Rack1?hep mice and littermate controls were subjected to quantitative RT-PCR (B), IB (C), and IHC (E, scale bar, 50 µm) to examine CXCL1 and S100A9 expression. (D) 48 h after transfection with siRNA against murine RACK1 or non-targeting control (NC) siRNA, BNL CL.2 cells were subjected to quantitative RT-PCR. (F, G) Liver mononuclear cells were purified from 12-week-old Rack1?hep mice and littermate controls, followed by flow cytometry analysis of Gr1+CD11b+ subset in CD45+ cells (F) or Ly6GhiLy6Clo and Ly6GloLy6Chi subsets in CD45+CD11b+F4/80- cells (G). Representative plots (left) and statistical data (right) are shown. (H) CD11b+F4/80-Ly6GhiLy6Clo myeloid cells were sorted from livers of Rack1?hep mice before and 2 h after LPS/GalN challenge. CFSE-labeled CD4+ T cells from spleens of Rack1F/F mice were cultured with or without two-fold number of these myeloid cells in the presence (w/) or absence (w/o) of Dynabeads Mouse T-Activator CD3/CD28. Proliferation was assessed by flow-cytometric analysis of CFSE dilution 72 h later. (I) CD3+ T cells sorted from livers of Rack1F/F mice were stimulated with 1 µg/mL phorbol myristate acetate (PMA) and 1 µM ionomycin (Ino) for 4 h in the presence or absence of the same number of CD11b+F4/80-Ly6GhiLy6Clo myeloid cells sorted from livers of Rack1?hep mice. IFN-?, TNF-a, and IL-17A levels in the supernatants were then determined by ELISA. Error bars indicate mean ± SD. The n-value information is shown in the corresponding panel. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.